Il-7r-alpha specific antibodies for treating acute lymphoblastic leukemia

ABSTRACT

Antibodies and antigen binding fragments that specifically bind to IL-7Rα are disclosed. Nucleic acids encoding the antibodies and antigen binding fragments, and vectors including the nucleic acid molecules are also provided. Methods for detecting a ca cancer or a cell that expresses IL-7Rα using the antibodies and antigen binding fragments are disclosed, as is the use of the antibodies and antigen binding fragments to prevent and/or treat a subject with a cancer that expresses IL-7Rα, such as acute lymphoblastic leukemia.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Provisional Application No.62/238,612, filed Oct. 7, 2015, which is incorporated by reference inits entirety.

FIELD OF THE DISCLOSURE

This relates to monoclonal antibodies and antigen binding fragments thatspecifically bind to the α chain of the interleukin 7 receptor (IL-7Rα)and their use, for example, in methods of treating a subject with acutelymphoblastic leukemia (ALL), such as T cell ALL (T-ALL).

BACKGROUND

Acute lymphoblastic leukemia (ALL) is the most common cancer in children(with approximately 3250 new cases per year in the United States).Typically, ALL is caused by over-proliferation of immature T cells(T-ALL) or immature B cells (B-ALL). Although treatment for ALL hasimproved dramatically in recent decades, about 20% of ALL cases are notcured. Accordingly, ALL remains a leading cause of death in children.Further, current therapies for pediatric ALL in growing children isextremely toxic, for example causing cognitive impairment due to thetoxicity of chemotherapy on the developing brain. ALL can also occur inadults, and adult ALL has a far less favorable prognosis than pediatricALL. Thus, there exists a need for new therapies for ALL, particularlyfor targeted therapies that have reduced cytotoxicity compared tostandard chemotherapeutic regimens.

SUMMARY

Isolated monoclonal antibodies and antigen binding fragments thatspecifically bind to the extracellular domain of IL-7Rα are providedherein. The disclosed antibodies and antigen binding fragments areuseful, for example, for treating or preventing ALL (such as T-ALL) in asubject. In some embodiments, the antibody or antigen binding fragmentspecifically binds to an IL-7Rα extracellular domain and comprises aheavy chain variable region (V_(H)) comprising a HCDR1, a HCDR2, and aHCDR3 of the V_(H) set forth as SEQ ID NO: 1 (4A10 V_(H)) and/or a lightchain variable region (V_(L)) comprising a LCDR1, a LCDR2, and a LCDR3of the V_(L) set forth as SEQ ID NO: 2 (4A10 V_(L)). In additionalembodiments, the antibody or antigen binding fragment comprises a V_(H)comprising a HCDR1, a HCDR2, and a HCDR3 of the V_(H) set forth as SEQID NO: 3 (2B8 V_(H)) and/or a V_(L) comprising a LCDR1, a LCDR2, and aLCDR3 of the V_(L) set forth as SEQ ID NO: 4 (2B8 V_(H)). In severalembodiments, the disclosed antibodies and antigen binding fragments canspecifically bind to the IL-7Rα extracellular domain expressed on a cellsurface.

The disclosed 4A10 and 2B8 antibodies are non-naturally occurringantibodies that were isolated from a laboratory screen of mousehybridoma cell lines. Chimeric forms of the 4A10 and 2B8 antibodies areprovided, for example, that include the heavy and light chain variableregions of the 4A10 or 2B8 antibody and human IgG (such as human IgG1)constant regions. In several embodiments, the disclosed antibodies andantigen binding fragments (for example chimeric forms of the disclosedantibodies or antigen binding fragments that include human IgG1 constantregions) can mediate antibody-dependent cell cytotoxicity (ADCC) againstcells with cell-surface expression of IL-7Rα.

Also disclosed are compositions including the antibodies and antigenbinding fragments, nucleic acids encoding the antibodies and antigenbinding fragments, expression vectors comprising the nucleic acids, andisolated host cells that comprise the nucleic acids.

The disclosed antibodies potently reduce proliferation of cancer cellsin an accepted in vivo model of ALL. Accordingly, a method is disclosedfor treating or inhibiting ALL (such as T-ALL) in a subject. The methodsinclude administering a therapeutically effective amount of one or moreof the disclosed antibodies, antigen binding fragments, nucleic acidmolecules, vectors, or compositions, to the subject, for example to asubject at risk of or having an IL-7Rα-positive cancer, such as ALL, forexample T-ALL or B-ALL. In some embodiments, the method comprisesadministration of a therapeutically effective amount of a combinationtherapy including administration of one or more of the disclosedIL-7Rα-specific antibodies, antigen binding fragments, nucleic acidmolecules, vectors, or compositions in combination with administrationof an additional agent, such as a CXCR4 antagonist (for example,AMD3100) to the subject, for example to a subject at risk of or havingan IL-7Rα-positive cancer, such as ALL, for example T-ALL or B-ALL.

The antibodies, antigen binding fragments, nucleic acid molecules,vectors, and compositions disclosed herein can be used for a variety ofadditional purposes, such as for detecting IL-7Rα expression on thesurface of a cell, diagnosing an IL-7Rα-positive cancer (such as T-ALL)in a subject, identifying a subject with ALL (such as T-ALL) that willrespond to therapy with a disclosed IL-7Rα antibody, or detecting IL-7Rαin a sample.

In additional embodiments, a method is provided for treating orpreventing an autoimmune disease in a subject, the method comprisingadministering a therapeutically effective amount of a disclosed IL-7Rαspecific antibody or antigen binding fragment to the subject.Non-limiting examples of autoimmune diseases that can be treated with adisclosed IL-7Rα specific antibody or antigen binding fragment includerheumatoid arthritis, type I diabetes, atopic dermatitis, multiplesclerosis, primary biliary cirrhosis, inflammatory bowel disease,sarcoidosis, or graft versus host disease.

The foregoing and other features and advantages of this disclosure willbecome more apparent from the following detailed description of severalembodiments which proceeds with reference to the accompanying figures.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a set of graphs illustrating prior findings that mutations inIL-7Rα are found in pediatric T-ALL patients. Three patient cohorts (theBoldrini, DCOG, and COALL patient cohorts) and the total are shown (seeZenatti et al., Nat. Genetics, 43:932-939, 2011).

FIG. 2 is a schematic diagram illustrating the prior finding concerningtypical somatic mutations in the IL-7Rα gene in T-ALL patients. Patient1 (P1) and patient 7 (P7) revealed cysteine insertions at the border ofthe extracellular and transmembrane regions of exon 6. The “P1” and “P7”mutations in the IL-7Rα gene include the mutations shown for the P1 andP7 patients, respectively.

FIG. 3 is a schematic diagram illustrating prior findings concerninggenetic mutations in the IL-7 pathway that can lead to T- or B-cellproliferation in ALL. ALL involves the IL-7 receptor pathways. The IL-7pathway frequently drives T cell proliferation in T-ALL, whereas thethymic stromal lymphopoietin (TSLP) pathway frequently drives B cellproliferation in B-ALL. IL-7 acts on lymphocytes by binding with highaffinity to IL-7Rα, and then recruiting the common γc chain. Thisheterodimerization brings together the intracellular domains of IL-7Rαand γc and their associated kinases, Jak1 and Jak3, respectively (J1 andJ3 in the figure). TSLP acts on lymphocytes by binding with highaffinity to IL-7Rα, and then recruiting the TSLP receptor (TSLPR). Thisheterodimerization brings together the intracellular domains of IL-7Rαand TSLPR and their associated kinases, Jak1 and Jak2, respectively (J1and J2 in the figure). Jak1 and Jak3 (for the IL-7Rα/γc heterodimer) orJak1 and Jak2 (for the IL-7Rα/TSLPR heterodimer) then phosphorylate asite on the intracellular domain of IL-7Rα, which recruits Stat5b.Stat5b is then phosphorylated, inducing its dimerization anddissociation from IL-7Rα and translocation to the nucleus where itserves as a transcription factor, inducing genes involved in survivaland proliferation. Mutations at any point in the IL-7 or TSLP pathwaycan lead to inappropriate phosphorylation of Stat5b, and resultinglymphocyte proliferation. The P1 and P7 gain-of function mutations inIL-7Rα illustrated in FIG. 2 lead to aberrant IL-7Rα homodimerization,phosphorylation of Stat5b by Jak1, and resulting lymphocyteproliferation.

FIG. 4 is a set of graphs illustrating the binding kinetics of 4A10 and2B8 scFvs for binding to IL-7Rα extracellular domain. scFvs includingthe heavy and light chain variable regions of the 4A10 or 2B8 antibodywere prepared and assayed for binding to purified IL-7Rα ectodomainusing surface plasmon resonance. The resulting K_(D) values for 2B8 or4A10 binding to IL-7Rα are shown.

FIG. 5 is a graph illustrating surface plasmon resonance data showingthat the 4A10 and 2B8 scFvs bind to non-overlapping epitopes on theIL-7Rα extracellular domain. IL-7Rα ectodomain was coupled to the SPRsensor-chip and binding was assayed by injecting 100 μL (400 μM) of 4A10or 2B8 scFv to the cassette, followed by another 100 mL (400 mM) of 4A10or 2B8 scFv. The dashed line indicates the time point when the secondscFv solution was injected. An increased response was observed when 2B8injection was followed by a 4A10 injection, indicating that these twoscFvs bind to non-overlapping epitopes on IL-7Rα.

FIG. 6 is a set of graphs showing results of FACS binding assaysindicating that a chimeric antibody including the 4A10 heavy and lightchain variable regions and human IgG1 constant regions (4A10-hIgG1chimera) binds wild type and mutant IL-7Rα. The amino acid sequences ofthe heavy and light chains of the 4A10-hIgG1 chimera are provided as SEQID NOs: 21 and 22, respectively. The 4A10-hIgG1 chimera was tested atvarious concentrations against BaF3 cells transfected with wild-type(WT) IL-7Rα, IL-7Rα including the P1 or P7 gain-of function mutation, ora control (pMIG vector).

FIG. 7 is a graph showing that the 4A10-hIgG1 chimera binds to humanT-ALL cells. A human T-ALL patient sample was expanded inimmunodeficient (NSG) mice. Spleen cells were harvested from the mice,and assayed for 4A10-hIgG1 chimera binding using FACS. The 4A10 antibodydoes not bind to mouse T- or B-cells. Accordingly, 4A10 binding to thehuman T-ALL cell expanded in the immunodeficient mice and harvested frommouse spleen indicates that this antibody binds to cell-surface IL-7Rαon the human T-ALL cells.

FIG. 8 illustrates that the 4A10-hIgG1 chimera mediates Natural Killer(NK)-cell ADCC against BaF3 cells expressing human IL-7Rα. BaF3 cellsare a murine B cell line that does not express human IL-7Rα and is notbound by the 4A10 antibody. The cells were incubated with NK cellsisolated from human blood at the indicated effector:target (E:T) ratiosand with the 4A10-hIgG1 chimera (10 μg/ml). Release of lactatedehydrogenase (LDH) was measured to evaluate cell lysis (cytotoxicity).

FIG. 9 illustrates that the 4A10-hIgG1 chimera mediates NK-cell ADCCagainst D1 cells expressing human mutant IL-7Rα from a T-ALL patient. D1cells are a murine T cell line that does not express human IL-7Rα and isnot bound by the 4A10 antibody. The cells were incubated with NK cellsisolated from human blood at the indicated E:T ratios and with the4A10-hIgG1 chimera (10 μg/ml). Release of LDH was measured to evaluatecell lysis (cytotoxicity).

FIG. 10 illustrates that the 4A10-hIgG1 chimera mediates NK-cell ADCCagainst human DND41 T-ALL cells that express IL-7Rα with again-of-function mutation. The cells were incubated with NK cellsisolated from human blood at the indicated E:T ratios and with the4A10-hIgG1 chimera (10 μg/ml). Release of LDH was measured to evaluatecell lysis (cytotoxicity).

FIG. 11 illustrates that the 4A10-hIgG1 chimera mediates NK-cell ADCCagainst normal human T cells. The cells were incubated with NK cellsisolated from human blood at the indicated E:T ratios and with the4A10-hIgG1 chimera (10 μg/ml). Release of LDH was measured to evaluatecell lysis (cytotoxicity).

FIGS. 12A-12F illustrate that the 4A10-hIgG1 chimera reducesproliferation of D1 cells expressing human IL-7Rα with a gain-offunction mutation in an in vivo assay, and promotes survival of miceinoculated with such cells. D1 cells transfected with mutant (P1) IL-7Rαand GFP were injected intravenously into Rag1^(−/−) mice. The followingday, 4A10-hIgG1 chimera (50-400 μg) or PBS (control) was administeredintravenously to the mice. Blood and tissue were sampled at various timepoints post-inoculation, and the overall survival of the mice was alsoevaluated (FIGS. 12B-12F).

FIGS. 13A-13D illustrate that administration of the 4A10-hIgG1 chimerareduces proliferation of human T-ALL cells expressing WT IL-7Rα in an invivo assay. (13A) Human T-ALL cells expressing WT IL-7Rα were injectedintravenously into immunodeficient NSG mice which lack NK cells as wellas T and B cells. 4A10-hIgG1 chimera (400 μg) or PBS (control) wereadministered intravenously at weekly intervals to the mice totaling fiveinjections. Blood and tissue were sampled at various time pointspost-inoculation and evaluated by IL-7Rα human CD4 staining to assayT-ALL cell proliferation (FIGS. 13B-13D).

FIGS. 14A-14F illustrate that administration of the 4A10-hIgG1 chimerareduces proliferation of human T-ALL cells expressing WT IL-7Rα in an invivo assay using NOD.SCID mice which have NK cells but lack T and Bcells. (14A) Human T-ALL cells isolated from a patient and expressing WTIL-7Rα were injected intravenously into NOD.SCID mice. 4A10-hIgG1chimera (250 μg) or PBS (control) were administered intravenously atweekly intervals to the mice totaling five injections. Blood and tissuewere sampled at various time points post-inoculation and evaluated byhuman CD45 staining to assay human T-ALL cell proliferation in blood(FIG. 14B), liver (FIG. 14C), lung (FIG. 14D), and bone marrow (FIG.14E). Mouse survival is show in FIG. 14F.

FIG. 15 is a set of graphs showing that anti-IL-7Rα antibodies caninhibit IL-7 signaling in IL-7Rα positive T-ALL cells. IL-7Rα positiveT-ALL cells were treated with IL-7 and the indicated antibodies, andevaluated for pSTAT-5 induction, which is induced following IL-7activation of IL-7R.

FIG. 16 shows a set of graphs illustrating the synergy of combinationtherapy including the CXCR4 antagonist AMD3100 and the 4A10-hIgG1chimera against T-ALL in bone marrow of NSG mice which lack NK cells.

SEQUENCE LISTING

The nucleic and amino acid sequences are shown using standard letterabbreviations for nucleotide bases, and three letter code for aminoacids, as defined in 37 C.F.R. 1.822. Only one strand of each nucleicacid sequence is shown, but the complementary strand is understood asincluded by any reference to the displayed strand. The Sequence Listingis submitted as an ASCII text file in the form of the file named“Sequence.txt” (˜52 kb), which was created on Sep. 29, 2016 which isincorporated by reference herein. In the accompanying sequence listing:

Bold highlighting in SEQ ID NOs: 1-4 indicates kabat CDR sequences. Boldhighlighting in SEQ ID NOs: 17-24 indicates constant region sequences.

SEQ ID NO: 1 is the amino acid sequence of the V_(H) of the 4A10 mAb.QVQLQQPGAELVMPGASVKLSCKASGYTFTSYWMHWVKQRPGEGLEWIGEIDPSDSYTNDNQKFKGKATLTVDKSSSTAYMQLSSLTSEDSAVYYCARRLYSNSYYYAMDYWGQGTSVTVSSSEQ ID NO: 2 is the amino acid sequence of the V_(L) of the 4A10 mAb.DIQMTQSPSSLSASLGGKVTITCKASQDIKKYIAWYQHKPGKGPRLLIHYTSTLQPGIPSRFSGSGSGRDYSFSISNLEPVDIATYYCLQYDNLLTFGAGTKLELKSEQ ID NO: 3 is the amino acid sequence of the V_(H) of the 2B8 mAb.EVQLQQSGPELVKPGASVKMSCKASGYTFSDYYMHWVKQSHGKSLEWIGYIYPDNGGNGYNQKFKGKATLTVDKSSSTVYMELRSLTSEDSALYYCARGTYYDGSYFDYWGQGTTLTVSSSEQ ID NO: 4 is the amino acid sequence of the V_(L) of the 2B8 mAb.DIVMTQSHKFMSTLVGDRVSITCKASQDVSTTVAWYQQKPGQSPKLLIYSASYRYTGVPDRFTGSGSGTDFTFTISSVQAEDLAVYYCQQHYSIPRTFGGGTKLEIKSEQ ID NOs: 5-16 are amino acid sequences of the kabat CDRs of the4A10 and 2B8 antibodies.SEQ ID NO: 17 is the amino acid sequence of the heavy chain of the4A10 mAb.QVQLQQPGAELVMPGASVKLSCKASGYTFTSYWMHWVKQRPGEGLEWIGEIDPSDSYTNDNQKFKGKATLTVDKSSSTAYMQLSSLTSEDSAVYYCARRLYSNSYYYAMDYWGQGTSVTVSSAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSTWPSQTVTCNVAHPASSTKVDKKIVPRDCGCKPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTKPREEQINSTFRSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTISKTKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITNFFPEDITVEWQWNGQPAENYKNTQPIMDTDGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGKSEQ ID NO: 18 is the amino acid sequence of the light chain of the4A10 mAb.DIQMTQSPSSLSASLGGKVTITCKASQDIKKYIAWYQHKPGKGPRLLIHYTSTLQPGIPSRFSGSGSGRDYSFSISNLEPVDIATYYCLQYDNLLTFGAGTKLELKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNEYPRDINVKWKIDGSERQNGVLNSWTDQDSKDSTYNMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC SEQ ID NO: 19 is the amino acid sequence of the heavy chain of the2B8 mAb.EVQLQQSGPELVKPGASVKMSCKASGYTFSDYYMHWVKQSHGKSLEWIGYIYPDNGGNGYNQKFKGKATLTVDKSSSTVYMELRSLTSEDSALYYCARGTYYDGSYFDYWGQGTTLTVSSAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSTWPSQTVTCNVAHPASSTKVDKKIVPRDCGCKPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTKPREEQINSTFRSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTISKTKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITNFFPEDITVEWQWNGQPAENYKNTQPIMDTDGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGKSEQ ID NO: 20 is the amino acid sequence of the light chain of the2B8 mAb.DIVMTQSHKFMSTLVGDRVSITCKASQDVSTTVAWYQQKPGQSPKLLIYSASYRYTGVPDRFTGSGSGTDFTFTISSVQAEDLAVYYCQQHYSIPRTFGGGTKLEIKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPRDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC SEQ ID NO: 21 is the amino acid sequence of a chimeric heavy chainincluding the 4A10 V_(H) and a human IgG1 constant region.QVQLQQPGAELVMPGASVKLSCKASGYTFTSYWMHWVKQRPGEGLEWIGEIDPSDSYTNDNQKFKGKATLTVDKSSSTAYMQLSSLTSEDSAVYYCARRLYSNSYYYAMDYWGQGTSVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKSEQ ID NO: 22 is the amino acid sequence of a chimeric light chainincluding the 4A10 V_(L) and a human IgG1 constant region.DIQMTQSPSSLSASLGGKVTITCKASQDIKKYIAWYQHKPGKGPRLLIHYTSTLQPGIPSRFSGSGSGRDYSFSISNLEPVDIATYYCLQYDNLLTFGAGTKLELKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO: 23 is the amino acid sequence of a chimeric heavy chainincluding the 2B8 V_(H) and a human IgG1 constant region.EVQLQQSGPELVKPGASVKMSCKASGYTFSDYYMHWVKQSHGKSLEWIGYIYPDNGGNGYNQKFKGKATLTVDKSSSTVYMELRSLTSEDSALYYCARGTYYDGSYFDYWGQGTTLTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKSEQ ID NO: 24 is the amino acid sequence of a chimeric light chainincluding the 2B8 V_(L) and a human IgG1 constant region.DIVMTQSHKFMSTLVGDRVSITCKASQDVSTTVAWYQQKPGQSPKLLIYSASYRYTGVPDRFTGSGSGTDFTFTISSVQAEDLAVYYCQQHYSIPRTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECSEQ ID NO: 25 is an exemplary nucleic acid sequence encoding the V_(H)of the 4A10 mAb.caggtccaactgcagcagcctggggctgagcttgtgatgcctggggcttcagtgaagctgtcctgcaaggcttctggctacaccttcaccagctactggatgcactgggtgaagcagaggcctggagaaggccttgagtggatcggagagattgatccttctgatagttatactaacgacaatcaaaagttcaagggcaaggccacattgactgtagacaaatcctccagcacagcctacatgcagctcagcagcctgacatctgaggactctgcggtctattactgtgcaagaaggctctatagtaactcttattactatgctatggactactggggtcaaggaacctcagtcaccgtctcctcaSEQ ID NO: 26 is an exemplary nucleic acid sequence encoding the V_(L)of the 4A10 mAb.gacatccagatgacacagtctccatcctcactgtctgcatctctgggaggcaaagtcaccatcacttgcaaggcaagccaagacattaagaagtatatagcttggtaccaacacaagcctggaaaaggtcctaggctgctcatacattacacatctacattacagccaggcatcccatcaaggttcagtggaagtgggtctgggagagattattccttcagcatcagcaacctggagcctgtggatattgcaacttattattgtctgcagtatgataatcttctcacattcggtgctgggaccaagctggagctgaaaSEQ ID NO: 27 is an exemplary nucleic acid sequence encoding the V_(H)of the 2B8 mAb.gaggtccagctgcaacagtctggacctgagttggtgaagcctggggcttcagtgaagatgtcctgcaaggcttctggctacacattcagtgactactacatgcactgggtgaagcagagccatggaaagagccttgagtggattggatatatttatcctgacaatggtggtaatggctacaaccagaagttcaagggcaaggccacattgactgtagacaagtcctccagcacagtctacatggagctccgcagcctgacatctgaggactctgcactctattactgtgcaagagggacctactatgatggttcctactttgactactggggccaaggcaccactctcacagtctcctcaSEQ ID NO: 28 is an exemplary nucleic acid sequence encoding the V_(L)of the 2B8 mAb.gacattgtgatgacccagtctcacaaattcatgtccacattagtaggagacagggtcagcatcacctgcaaggccagtcaggatgtgagtactactgtagcctggtatcaacagaaaccaggacaatctcctaaactactgatttactcggcatcctaccggtacactggagtccctgatcgcttcactggcagtggatctgggacggatttcactttcaccatcagcagtgtgcaggctgaagacctggcagtttattactgtcaacaacattatagtattcctcggacgttcggtggaggcaccaagctggaaatcaaaSEQ ID NO: 29 is an exemplary nucleic acid sequence encoding theheavy chain of the 4A10 mAb.caggtccaactgcagcagcctggggctgagcttgtgatgcctggggcttcagtgaagctgtcctgcaaggcttctggctacaccttcaccagctactggatgcactgggtgaagcagaggcctggagaaggccttgagtggatcggagagattgatccttctgatagttatactaacgacaatcaaaagttcaagggcaaggccacattgactgtagacaaatcctccagcacagcctacatgcagctcagcagcctgacatctgaggactctgcggtctattactgtgcaagaaggctctatagtaactcttattactatgctatggactactggggtcaaggaacctcagtcaccgtctcctcagccaaaacgacacccccatctgtctatccactggcccctggatctgctgcccaaactaactccatggtgaccctgggatgcctggtcaagggctatttccctgagccagtgacagtgacctggaactctggatccctgtccagcggtgtgcacaccttcccagctgtcctgcagtctgacctctacactctgagcagctcagtgactgtcccctccagcacctggcccagccagaccgtcacctgcaacgttgcccacccggccagcagcaccaaggtggacaagaaaattgtgcccagggattgtggttgtaagccttgcatatgtacagtcccagaagtatcatctgtcttcatcttccccccaaagcccaaggatgtgctcaccattactctgactcctaaggtcacgtgtgttgtggtagacatcagcaaggatgatcccgaggtccagttcagctggtttgtagatgatgtggaggtgcacacagctcagacgaaaccccgggaggagcagatcaacagcactttccgttcagtcagtgaacttcccatcatgcaccaggactggctcaatggcaaggagttcaaatgcagggtcaacagtgcagctttccctgcccccatcgagaaaaccatctccaaaaccaaaggcagaccgaaggctccacaggtgtacaccattccacctcccaaggagcagatggccaaggataaagtcagtctgacctgcatgataacaaacttcttccctgaagacattactgtggagtggcagtggaatgggcagccagcggagaactacaagaacactcagcccatcatggacacagatggctcttacttcgtctacagcaagctcaatgtgcagaagagcaactgggaggcaggaaatactttcacctgctctgtgttacatgagggcctgcacaaccaccatactgagaagagcctctcccactctcctggtaaaSEQ ID NO: 30 is an exemplary nucleic acid sequence encoding the light chain of the 4A10 mAb.gacatccagatgacacagtctccatcctcactgtctgcatctctgggaggcaaagtcaccatcacttgcaaggcaagccaagacattaagaagtatatagcttggtaccaacacaagcctggaaaaggtcctaggctgctcatacattacacatctacattacagccaggcatcccatcaaggttcagtggaagtgggtctgggagagattattccttcagcatcagcaacctggagcctgtggatattgcaacttattattgtctgcagtatgataatcttctcacattcggtgctgggaccaagctggagctgaaacgggctgatgctgcaccaactgtatccatcttcccaccatccagtgagcagttaacatctggaggtgcctcagtcgtgtgcttcttgaacaacttctaccccagagacatcaatgtcaagtggaagattgatggcagtgaacgacaaaatggtgtcctgaacagttggactgatcaggacagcaaagacagcacctacaacatgagcagcaccctcacattgaccaaggacgagtatgaacgacataacagctatacctgtgaggccactcacaagacatcaacttcacccatcgtcaagagcttcaacaggaatgagtgtSEQ ID NO: 31 is an exemplary nucleic acid sequence encoding the heavy chain of the 2B8 mAb.gaggtccagctgcaacagtctggacctgagttggtgaagcctggggcttcagtgaagatgtcctgcaaggcttctggctacacattcagtgactactacatgcactgggtgaagcagagccatggaaagagccttgagtggattggatatatttatcctgacaatggtggtaatggctacaaccagaagttcaagggcaaggccacattgactgtagacaagtcctccagcacagtctacatggagctccgcagcctgacatctgaggactctgcactctattactgtgcaagagggacctactatgatggttcctactttgactactggggccaaggcaccactctcacagtctcctcagccaaaacgacacccccatctgtctatccactggcccctggatctgctgcccaaactaactccatggtgaccctgggatgcctggtcaagggctatttccctgagccagtgacagtgacctggaactctggatccctgtccagcggtgtgcacaccttcccagctgtcctgcagtctgacctctacactctgagcagctcagtgactgtcccctccagcacctggcccagccagaccgtcacctgcaacgttgcccacccggccagcagcaccaaggtggacaagaaaattgtgcccagggattgtggttgtaagccttgcatatgtacagtcccagaagtatcatctgtcttcatcttccccccaaagcccaaggatgtgctcaccattactctgactcctaaggtcacgtgtgttgtggtagacatcagcaaggatgatcccgaggtccagttcagctggtttgtagatgatgtggaggtgcacacagctcagacgaaaccccgggaggagcagatcaacagcactttccgttcagtcagtgaacttcccatcatgcaccaggactggctcaatggcaaggagttcaaatgcagggtcaacagtgcagctttccctgcccccatcgagaaaaccatctccaaaaccaaaggcagaccgaaggctccacaggtgtacaccattccacctcccaaggagcagatggccaaggataaagtcagtctgacctgcatgataacaaacttcttccctgaagacattactgtggagtggcagtggaatgggcagccagcggagaactacaagaacactcagcccatcatggacacagatggctcttacttcgtctacagcaagctcaatgtgcagaagagcaactgggaggcaggaaatactttcacctgctctgtgttacatgagggcctgcacaaccaccatactgagaagagcctctcccactctcctggtaaa SEQ ID NO: 32 is an exemplary nucleic acid sequence encoding the light chain of the 2B8 mAb.gacattgtgatgacccagtctcacaaattcatgtccacattagtaggagacagggtcagcatcacctgcaaggccagtcaggatgtgagtactactgtagcctggtatcaacagaaaccaggacaatctcctaaactactgatttactcggcatcctaccggtacactggagtccctgatcgcttcactggcagtggatctgggacggatttcactttcaccatcagcagtgtgcaggctgaagacctggcagtttattactgtcaacaacattatagtattcctcggacgttcggtggaggcaccaagctggaaatcaaacgggctgatgctgcaccaactgtatccatcttcccaccatccagtgagcagttaacatctggaggtgcctcagtcgtgtgcttcttgaacaacttctaccccagagacatcaatgtcaagtggaagattgatggcagtgaacgacaaaatggtgtcctgaacagttggactgatcaggacagcaaagacagcacctacagcatgagcagcaccctcacattgaccaaggacgagtatgaacgacataacagctatacctgtgaggccactcacaagacatcaacttcacccatcgtcaagagcttcaacaggaatgagtgtSEQ ID NO: 33 is an exemplary nucleic acid sequence encoding a chimeric heavy chain including the 4A10 V_(H) and a human IgG1 constantregion.caggtccagctgcagcagcccggagccgaactggtcatgcccggcgccagcgtgaagctgtcctgcaaggcttctggctataccttcacatcctactggatgcactgggtgaagcagagacccggagagggactggagtggatcggcgagatcgacccatccgattcttataccaacgacaatcagaagtttaagggcaaggccaccctgacagtggataagagctcctctaccgcttatatgcagctgagctccctgacaagcgaggactccgccgtgtactattgcgctcggaggctgtatagcaactcctactattacgccatggattactggggccagggcaccagcgtgacagtgtctagcgccagcaccaagggcccttccgtgttcccactggctccctcctctaaatctaccagcggaggaacagccgctctgggatgtctggtgaaggactacttcccagagcccgtgacagtgtcttggaacagcggcgccctgacctccggcgtgcacacatttcctgctgtgctgcagagctccggcctgtattctctgtctagcgtggtgaccgtgccatcctctagcctgggcacccagacatacatctgcaacgtgaatcacaagccatccaatacaaaggtggacaagaaggtcgagcccaagtcttgtgataagacccacacatgccccccttgtcctgctccagagctgctgggaggaccttccgtgttcctgtttccacccaaacctaaggacaccctgatgatcagccggaccccagaggtgacatgcgtggtggtggacgtgtcccacgaggatcccgaggtgaagtttaactggtacgtcgatggcgtggaggtgcacaatgctaagaccaagcccagagaggagcagtataactctacctaccgggtggtgagcgtgctgacagtgctgcaccaggactggctgaacggcaaggagtacaagtgcaaggtgtctaataaggccctgcccgctcccatcgagaagaccatcagcaaggccaagggccagcctagggagccacaggtgtatacactgcctccatctagagacgagctgaccaagaaccaggtgagcctgacatgtctggtgaagggcttctaccccagcgatatcgccgtggagtgggagtccaatggccagcctgagaacaattataagaccacaccccctgtgctggactccgatggctctttctttctgtactccaagctgaccgtggataagtctaggtggcagcagggcaacgtgttcagctgttctgtgatgcacgaagctctgcataatcactacacccagaaaagcctgtccctgtcacctggtaaaSEQ ID NO: 34 is an exemplary nucleic acid sequence encoding a chimeric light chain including the 4A10 V_(L) and a human IgG1 constantregion.gatattcagatgactcagtccccttcttcactgtctgccagcctgggcggcaaggtgaccatcacatgcaaggcctcccaggacatcaagaagtacatcgcttggtatcagcacaagcctggcaagggcccacggctgctgatccactacacctctacactgcagccaggcatccccagccggttctccggaagcggaagcggaagggattactccttttctatcagcaacctggagcccgtggacatcgccacctactattgcctgcagtatgataatctgctgaccttcggcgctggcacaaagctggagctgaagagaaccgtggccgctcccagcgtgttcatctttcccccttccgacgagcagctgaagtccggcacagcttctgtggtgtgcctgctgaacaacttctaccctcgggaggccaaggtgcagtggaaggtggataacgctctgcagtccggcaattctcaggagagcgtgaccgagcaggactccaaggattctacatatagcctgagctccaccctgacactgtctaaggccgactacgagaagcacaaggtgtatgcttgtgaagtgactcatcagggtctgtcatcacccgtgactaaatcattcaatagaggcgaatgc SEQ ID NO: 35 is an exemplary nucleic acid sequence encoding achimeric heavy chain including the 2B8 V_(H) and a human IgG1 constantregion.gaagtccagctgcagcagtctggtcctgaactggtgaagcctggcgcctccgtgaagatgtcttgcaaggctagcggctacaccttcagcgactactatatgcactgggtgaagcagtcccacggcaagtctctggagtggatcggctacatctatccagataacggcggcaatggctacaaccagaagtttaagggcaaggccaccctgacagtggacaagagctcctctaccgtgtatatggagctgaggagcctgacatccgaggattctgccctgtactattgcgctagaggcacctactatgacggctcctacttcgattattggggccagggcaccacactgaccgtgagctccgccagcacaaagggcccctccgtgtttccactggctccctcttccaagtctaccagcggaggaacagccgctctgggatgtctggtgaaggactacttcccagagcccgtgaccgtgagctggaacagcggcgccctgacctccggcgtgcacacatttccagctgtgctgcagtcctctggcctgtactctctgagctccgtggtgaccgtgccctctagctccctgggcacccagacatatatctgcaacgtgaatcacaagccatccaacacaaaggtggacaagaaggtcgagcccaagtcttgtgataagacccacacatgccccccttgtcctgctcccgagctgctgggaggacctagcgtgttcctgtttccacccaagcctaaggacaccctgatgatcagccggacccccgaggtgacatgcgtggtggtggacgtgtcccacgaggatcctgaggtgaagttcaattggtatgtcgatggcgtggaggtgcacaacgctaagacaaagcctcgggaggagcagtacaattctacctatagggtggtgagcgtgctgacagtgctgcaccaggactggctcaatggcaaggagtataagtgcaaggtgtctaacaaggccctgcccgctcctatcgagaagaccatcagcaaggccaagggccagcctagagagccacaggtgtacacactgcctccatctcgggacgagctgaccaagaatcaggtgagcctgacatgtctggtgaagggcttctatcctagcgatatcgccgtggagtgggagtccaacggccagccagagaacaattacaagaccacaccccctgtgctggactctgatggcagcttctttctgtattccaagctgaccgtggataagtctaggtggcagcagggcaacgtgttttcctgttctgtgatgcacgaagccctgcataatcactatactcagaaatccctgtcactgtcacctggtaaaSEQ ID NO: 36 is an exemplary nucleic acid sequence encoding a chimeric light chain including the 2B8 V_(L) and a human IgG1 constantregion.gacattgtgatgactcagtcccataaattcatgtctaccctggtgggcgaccgggtgagcatcacatgcaaggcctctcaggatgtgagcaccacagtggcttggtaccagcagaagccaggccagtcccccaagctgctgatctattccgcctcttatcggtataccggagtgcctgacaggttcaccggaagcggatccggcacagatttcacctttacaatcagctccgtgcaggccgaggacctggccgtgtactattgccagcagcactactctatccctagaacctttggcggcggcacaaagctggagatcaagcggaccgtggccgctccaagcgtgttcatctttcccccttccgacgagcagctgaagtccggcacagcttctgtggtgtgcctgctgaacaatttctaccccagggaggccaaggtccagtggaaggtggataacgctctgcagtctggcaatagccaggagtccgtgaccgagcaggactctaaggatagcacatattccctgtctagcaccctgacactgagcaaggccgattacgagaagcacaaggtgtatgcttgtgaagtcactcatcagggtctgtcttcacctgtcactaagtcttttaaccgaggcgaatgcSEQ ID NOs: 37-46 are amino acid sequences concerning chimericantigen receptors. SEQ ID NOs: 47-49 are peptide linker sequences.

DETAILED DESCRIPTION I. ABBREVIATIONS

ADC antibody-drug conjugate

ADCC antibody-dependent cellular cytotoxicity

ALL acute lymphoblastic leukemia

CAR chimeric antigen receptor

CD3 cluster of differentiation 3 T cell coreceptor

CDR complementarity determining region

CXC4 C-X-C chemokine receptor type 4

E:T effector:target

HCDR heavy chain complementarity determining region

hIgG human immunoglobulin G

IgG immunoglobulin G

IL-7 interleukin-7

IL-7Rα interleukin-7 receptor α

LCDR light chain complementarity determining region

LDH lactate dehydrogenase

NK natural killer

scFv single chain antibody

T-ALL T cell derived acute lymphoblastic leukemia

B-ALL B cell derived acute lymphoblastic leukemia

TSLP thymic stromal lymphopoietin

V_(H) heavy chain variable region

V_(L) light chain variable region

II. SUMMARY OF TERMS

Unless otherwise noted, technical terms are used according toconventional usage. Definitions of common terms in molecular biology maybe found in Benjamin Lewin, Genes X, published by Jones & BartlettPublishers, 2009; and Meyers et al. (eds.), The Encyclopedia of CellBiology and Molecular Medicine, published by Wiley-VCH in 16 volumes,2008; and other similar references.

As used herein, the singular forms “a,” “an,” and “the,” refer to boththe singular as well as plural, unless the context clearly indicatesotherwise. For example, the term “an antigen” includes single or pluralantigens and can be considered equivalent to the phrase “at least oneantigen.” As used herein, the term “comprises” means “includes.” It isfurther to be understood that any and all base sizes or amino acidsizes, and all molecular weight or molecular mass values, given fornucleic acids or polypeptides are approximate, and are provided fordescriptive purposes, unless otherwise indicated. Although many methodsand materials similar or equivalent to those described herein can beused, particular suitable methods and materials are described herein. Incase of conflict, the present specification, including explanations ofterms, will control. In addition, the materials, methods, and examplesare illustrative only and not intended to be limiting. To facilitatereview of the various embodiments, the following explanations of termsare provided:

Acute Lymphoblastic Leukemia (ALL): An acute leukemia involving theoverproduction and accumulation of cancerous lymphoblasts (immature B-and/or T cells). ALL is the most common cancer in children. Treatmentfor ALL has improved dramatically in recent decades, but there remainabout 20% of cases that are not cured and it remains a leading cause ofdeath in children. ALL can be subdivided into two broad groups:leukemias derived from immature T cells (T-ALL) and leukemias derivedfrom immature B cells (B-ALL). The majority of T-ALL and B-ALL cellsexpress IL-7Rα and respond to IL-7 in vitro, showing increased survivaland proliferation (see, e.g., Barata et al., Blood, 98:1524-1531, 2001;Touw et al., Blood, 75:2097-2101, 1990). Methods of diagnosing ALL in asubject, or diagnosing a subject with ALL as having T-ALL or B-ALL areknown (see, for example, Chiaretti et al., “Diagnosis andSubclassification of Acute Lymphoblastic Leukemia,” Mediterranean JHematol Infect Dis, 6(1): e2014073, 2014).

T-ALL can be caused by gain-of-function mutations in the IL-7Rα gene(see, Zenatti et al., Nat. Genet., 43:932-939, 2011). These mutationsare often insertions into exon 6 of IL-7Rα that encode cysteineresidues. Additionally, other gain-of-function mutations in the IL-7pathway that contribute to T cell or B cell proliferation are known(such as gain-of-function mutations in the genes encoding, Jak1, Jak3,Stat5b, Ras or AKT), as well as in B-ALL (such as gain-of-functionmutations in the genes encoding TSLPR or Jak2).

Administration: The introduction of a composition into a subject by achosen route. Administration can be local or systemic. For example, ifthe chosen route is intravenous, the composition is administered byintroducing the composition into a vein of the subject. Exemplary routesof administration include, but are not limited to, oral, injection (suchas subcutaneous, intramuscular, intradermal, intraperitoneal, andintravenous), sublingual, rectal, transdermal (for example, topical),intranasal, vaginal, and inhalation routes.

AMD3100: A CXCR4 antagonist currently sold by Genzyme Corporation as animmunostimulants used to mobilize hematopoietic stem cells from bonemarrow to the blood stream. AMD3100 is also known as Plerixafor andMozobil®. The chemical structure of AMD3100 is provided as:

Use and dosages for AMD3100 are known, see, e.g., Hummel et al., CurrOpin. Hematolog., 21(1):29-36, 2014 and Liu et al., Exp. Hematol. Opin.,5:19, 2016.

Amino acid substitution: The replacement of one amino acid in peptidewith a different amino acid.

Antibody: An immunoglobulin, antigen-binding fragment, or derivativethereof, that specifically binds and recognizes an analyte (antigen)such as IL-7Rα. The term “antibody” is used herein in the broadest senseand encompasses various antibody structures, including but not limitedto monoclonal antibodies, polyclonal antibodies, multispecificantibodies (e.g., bispecific antibodies), and antibody fragments, solong as they exhibit the desired antigen-binding activity.

Non-limiting examples of antibodies include, for example, intactimmunoglobulins and variants and fragments thereof known in the art thatretain binding affinity for the antigen. Examples of antibody fragmentsinclude but are not limited to Fv, Fab, Fab′, Fab′-SH, F(ab′)₂;diabodies; linear antibodies; single-chain antibody molecules (e.g.scFv); and multispecific antibodies formed from antibody fragments.Antibody fragments include antigen binding fragments either produced bythe modification of whole antibodies or those synthesized de novo usingrecombinant DNA methodologies (see, e.g., Kontermann and Dubel (Ed),Antibody Engineering, Vols. 1-2, 2^(nd) Ed., Springer Press, 2010).

A single-chain antibody (scFv) is a genetically engineered moleculecontaining the V_(H) and V_(L) domains of one or more antibody(ies)linked by a suitable polypeptide linker as a genetically fused singlechain molecule (see, for example, Bird et al., Science, 242:423-426,1988; Huston et al., Proc. Natl. Acad. Sci., 85:5879-5883, 1988; Ahmadet al., Clin. Dev. Immunol., 2012, doi:10.1155/2012/980250; Marbry,IDrugs, 13:543-549, 2010). The intramolecular orientation of theV_(H)-domain and the V_(L)-domain in a scFv, is typically not decisivefor scFvs. Thus, scFvs with both possible arrangements(V_(H)-domain-linker domain-V_(L)-domain; V_(L)-domain-linkerdomain-V_(H)-domain) may be used.

In a dsFv the V_(H) and V_(L) have been mutated to introduce a disulfidebond to stabilize the association of the chains. Diabodies also areincluded, which are bivalent, bispecific antibodies in which V_(H) andV_(L) domains are expressed on a single polypeptide chain, but using alinker that is too short to allow for pairing between the two domains onthe same chain, thereby forcing the domains to pair with complementarydomains of another chain and creating two antigen binding sites (see,for example, Holliger et al., Proc. Natl. Acad. Sci., 90:6444-6448,1993; Poljak et al., Structure, 2:1121-1123, 1994).

Antibodies also include genetically engineered forms such as chimericantibodies (such as humanized murine antibodies) and heteroconjugateantibodies (such as bispecific antibodies). See also, Pierce Catalog andHandbook, 1994-1995 (Pierce Chemical Co., Rockford, Ill.); Kuby, J.,Immunology, 3^(rd) Ed., W.H. Freeman & Co., New York, 1997.

An “antibody that binds to the same epitope” as a reference antibodyrefers to an antibody that blocks binding of the reference antibody toits antigen in a competition assay by 50% or more, and conversely, thereference antibody blocks binding of the antibody to its antigen in acompetition assay by 50% or more. Antibody competition assays are known,and an exemplary competition assay is provided herein.

An antibody may have one or more binding sites. If there is more thanone binding site, the binding sites may be identical to one another ormay be different. For instance, a naturally occurring immunoglobulin hastwo identical binding sites, a single-chain antibody or Fab fragment hasone binding site, while a bispecific or bifunctional antibody has twodifferent binding sites.

Typically, a naturally occurring immunoglobulin has heavy chains andlight chains interconnected by disulfide bonds. Immunoglobulin genesinclude the kappa, lambda, alpha, gamma, delta, epsilon and mu constantregion genes, as well as the myriad immunoglobulin variable domaingenes. There are two types of light chain, lambda (λ) and kappa (κ).There are five main heavy chain classes (or isotypes) which determinethe functional activity of an antibody molecule: IgM, IgD, IgG, IgA andIgE.

Each heavy and light chain contains a constant region (or constantdomain) and a variable region (or variable domain; see, e.g., Kindt etal. Kuby Immunology, 6^(th) ed., W.H. Freeman and Co., page 91 (2007).)In several embodiments, the V_(H) and V_(L) combine to specifically bindthe antigen. In additional embodiments, only the V_(H) is required. Forexample, naturally occurring camelid antibodies consisting of a heavychain only are functional and stable in the absence of light chain (see,e.g., Hamers-Casterman et al., Nature, 363:446-448, 1993; Sheriff etal., Nat. Struct. Biol., 3:733-736, 1996). References to “V_(H)” or “VH”refer to the variable region of an antibody heavy chain, including thatof an antigen binding fragment, such as Fv, scFv, dsFv or Fab.References to “V_(L)” or “VL” refer to the variable domain of anantibody light chain, including that of an Fv, scFv, dsFv or Fab.

The V_(H) and V_(L) contain a “framework” region interrupted by threehypervariable regions, also called “complementarity-determining regions”or “CDRs” (see, e.g., Kabat et al., Sequences of Proteins ofImmunological Interest, U.S. Department of Health and Human Services,1991). The sequences of the framework regions of different light orheavy chains are relatively conserved within a species. The frameworkregion of an antibody, that is the combined framework regions of theconstituent light and heavy chains, serves to position and align theCDRs in three-dimensional space.

The CDRs are primarily responsible for binding to an epitope of anantigen. The amino acid sequence boundaries of a given CDR can bereadily determined using any of a number of well-known schemes,including those described by Kabat et al. (“Sequences of Proteins ofImmunological Interest,” 5th Ed. Public Health Service, NationalInstitutes of Health, Bethesda, Md., 1991; “Kabat” numbering scheme),Al-Lazikani et al., (JMB 273,927-948, 1997; “Chothia” numbering scheme),and Lefranc et al. (“IMGT unique numbering for immunoglobulin and T cellreceptor variable domains and Ig superfamily V-like domains,” Dev. Comp.Immunol., 27:55-77, 2003; “IMGT” numbering scheme). The CDRs of eachchain are typically referred to as CDR1, CDR2, and CDR3 (from theN-terminus to C-terminus), and are also typically identified by thechain in which the particular CDR is located. Thus, a V_(H) CDR3 is theCDR3 from the V_(H) of the antibody in which it is found, whereas aV_(L) CDR1 is the CDR1 from the V_(L) of the antibody in which it isfound. Light chain CDRs are sometimes referred to as LCDR1, LCDR2, andLCDR3. Heavy chain CDRs are sometimes referred to as HCDR1, HCDR2, andHCDR3.

A “monoclonal antibody” is an antibody obtained from a population ofsubstantially homogeneous antibodies, that is, the individual antibodiescomprising the population are identical and/or bind the same epitope,except for possible variant antibodies, for example, containingnaturally occurring mutations or arising during production of amonoclonal antibody preparation, such variants generally being presentin minor amounts. In contrast to polyclonal antibody preparations, whichtypically include different antibodies directed against differentdeterminants (epitopes), each monoclonal antibody of a monoclonalantibody preparation is directed against a single determinant on anantigen. Thus, the modifier “monoclonal” indicates the character of theantibody as being obtained from a substantially homogeneous populationof antibodies, and is not to be construed as requiring production of theantibody by any particular method. For example, the monoclonalantibodies may be made by a variety of techniques, including but notlimited to the hybridoma method, recombinant DNA methods, phage-displaymethods, and methods utilizing transgenic animals containing all or partof the human immunoglobulin loci, such methods and other exemplarymethods for making monoclonal antibodies being described herein. In someexamples monoclonal antibodies are isolated from a subject. Monoclonalantibodies can have conservative amino acid substitutions, which havesubstantially no effect on antigen binding or other immunoglobulinfunctions. (See, for example, Harlow & Lane, Antibodies, A LaboratoryManual, 2^(nd) ed. Cold Spring Harbor Publications, New York (2013).)

A “humanized” antibody or antigen binding fragment includes a humanframework region and one or more CDRs from a non-human (such as a mouse,rat, or synthetic) antibody or antigen binding fragment. The non-humanantibody or antigen binding fragment providing the CDRs is termed a“donor,” and the human antibody or antigen binding fragment providingthe framework is termed an “acceptor.” In one embodiment, all the CDRsare from the donor immunoglobulin in a humanized immunoglobulin.Constant regions need not be present, but if they are, they can besubstantially identical to human immunoglobulin constant regions, suchas at least about 85-90%, such as about 95% or more identical. Hence,all parts of a humanized antibody or antigen binding fragment, exceptpossibly the CDRs, are substantially identical to corresponding parts ofnatural human antibody sequences.

A “chimeric antibody” is an antibody which includes sequences derivedfrom two different antibodies, and are typically of different species.In some examples, a chimeric antibody includes one or more CDRs and/orframework regions from one human antibody and CDRs and/or frameworkregions from another human antibody. In other embodiments, a chimericantibody can include the V_(H) and V_(L) regions of a mouse monoclonalantibody (such as the 4A10 or 2B8 antibody) and human constant regions,such as human IgG1 regions.

A “fully human antibody” or “human antibody” is an antibody, whichincludes sequences from (or derived from) the human genome, and does notinclude sequence from another species. In some embodiments, a humanantibody includes CDRs, framework regions, and (if present) an Fc regionfrom (or derived from) the human genome. Human antibodies can beidentified and isolated using technologies for creating antibodies basedon sequences derived from the human genome, for example by phage displayor using transgenic animals (see, e.g., Barbas et al. Phage display: ALaboratory Manuel. 1^(st) Ed. New York: Cold Spring Harbor LaboratoryPress, 2004. Print.; Lonberg, Nat. Biotech., 23: 1117-1125, 2005;Lonenberg, Curr. Opin. Immunol., 20:450-459, 2008)

Antibody-drug conjugate (ADC): A molecule that includes an antibody (orantigen-binding fragment of an antibody) conjugated to a drug, such as acytotoxic agent. ADCs can be used to specifically target a cytotoxicagent to cancer cells through specific binding of the antibody to atumor antigen expressed on the cell surface. Exemplary drugs for usewith ADCs include anti-microtubule agents (such as maytansinoids,auristatin E and auristatin F) and interstrand crosslinking agents(e.g., pyrrolobenzodiazepines or PDBs).

Autoimmune disease: A disorder in which the immune system produces animmune response (for example, a B cell or a T cell response) against anendogenous antigen, with consequent injury to tissues. For example,rheumatoid arthritis is an autoimmune disorder, as are Hashimoto'sthyroiditis, pernicious anemia, Addison's disease, type I diabetes,systemic lupus erythematosus, Atopic dermatitis, Inhalation Allergy,dermatomyositis, Sjogren's syndrome, dermatomyositis, lupuserythematosus, multiple sclerosis, myasthenia gravis, Reiter's syndrome,Primary Biliary Cirrhosis, Inflammatory bowel disease, and Grave'sdisease, among others.

Biological sample: A sample obtained from a subject. Biological samplesinclude all clinical samples useful for detection of disease (forexample, T-ALL) in subjects, including, but not limited to, cells,tissues, and bodily fluids, such as blood, derivatives and fractions ofblood (such as serum), cerebrospinal fluid; as well as biopsied orsurgically removed tissue, for example tissues that are unfixed, frozen,or fixed in formalin or paraffin. In a particular example, a biologicalsample is obtained from a subject having or suspected of having T-ALL.

Bispecific antibody: A recombinant molecule composed of two differentantigen binding domains that consequently binds to two differentantigenic epitopes. Bispecific antibodies include chemically orgenetically linked molecules of two antigen-binding domains. The antigenbinding domains can be linked using a linker. The antigen bindingdomains can be monoclonal antibodies, antigen-binding fragments (e.g.,Fab, scFv), or combinations thereof. A bispecific antibody can includeone or more constant domains, but does not necessarily include aconstant domain.

CD3 (Cluster of differentiation 3 T cell Co-receptor): A specificprotein complex including at least four polypeptide chains, which arenon-covalently associated with the T cell receptors on the surface of Tcells. The four polypeptide chains include two CD3-epsilon chains, aCD3-delta chain and a CD3-gamma chain. CD3 is present on both helper Tcells and cytotoxic T cells.

Chemotherapeutic agent: Any chemical agent with therapeutic usefulnessin the treatment of diseases characterized by abnormal cell growth. Forexample, chemotherapeutic agents can be useful for the treatment ofcancer, such as T-ALL or B-ALL, such as in combination therapy with oneor more of the disclosed IL-7Rα-specific antibodies. Particular examplesof chemotherapeutic agents that can be used include microtubule bindingagents, DNA intercalators or cross-linkers, DNA synthesis inhibitors,DNA and RNA transcription inhibitors, antibodies, enzymes, enzymeinhibitors, gene regulators, and angiogenesis inhibitors. In oneembodiment, a chemotherapeutic agent is a radioactive compound. Inanother embodiments, the chemotherapeutic agent is a CXCR4 antagonist,such as AMD3100. One of skill in the art can readily identify achemotherapeutic agent of use (see for example, Slapak and Kufe,Principles of Cancer Therapy, Chapter 86 in Harrison's Principles ofInternal Medicine, 14th edition; Perry et al., Chemotherapy, Ch. 17 inAbeloff, Clinical Oncology 2^(nd) ed., © 2000 Churchill Livingstone,Inc; Baltzer, L., Berkery, R. (eds): Oncology Pocket Guide toChemotherapy, 2nd ed. St. Louis, Mosby-Year Book, 1995; Fischer, D. S.,Knobf, M. F., Durivage, H. J. (eds): The Cancer Chemotherapy Handbook,4th ed. St. Louis, Mosby-Year Book, 1993; Chabner and Longo, CancerChemotherapy and Biotherapy: Principles and Practice (4th ed.).Philadelphia: Lippincott Willians & Wilkins, 2005; Skeel, Handbook ofCancer Chemotherapy (6th ed.). Lippincott Williams & Wilkins, 2003).Combination chemotherapy is the administration of more than one agent totreat cancer.

Chimeric Antigen Receptor (CAR): An engineered T cell receptor having anextracellular antibody-derived targeting domain (such as an scFv) joinedto one or more intracellular signaling domains of a T cell receptor. A“chimeric antigen receptor T cell” is a T cell expressing a CAR, and hasantigen specificity determined by the antibody-derived targeting domainof the CAR. Methods of making CARs are available (see, e.g., Park etal., Trends Biotechnol., 29:550-557, 2011; Grupp et al., N Engl J Med.,368:1509-1518, 2013; Han et al., J. Hematol Oncol., 6:47, 2013; PCTPubs. WO2012/079000, WO2013/059593; and U.S. Pub. 2012/0213783, each ofwhich is incorporated by reference herein in its entirety.)

Conditions sufficient to form an immune complex: Conditions which allowan antibody or antigen binding fragment thereof to bind to its cognateepitope to a detectably greater degree than, and/or to the substantialexclusion of, binding to substantially all other epitopes. Conditionssufficient to form an immune complex are dependent upon the format ofthe binding reaction and typically are those utilized in immunoassayprotocols or those conditions encountered in vivo. See Harlow & Lane(Antibodies, A Laboratory Manual, 2^(nd) ed. Cold Spring HarborPublications, New York, 2013) for a description of immunoassay formatsand conditions. The conditions employed in the methods are“physiological conditions” which include reference to conditions (e.g.,temperature, osmolarity, pH) that are typical inside a living mammal ora mammalian cell. While it is recognized that some organs are subject toextreme conditions, the intra-organismal and intracellular environmentnormally lies around pH 7 (e.g., from pH 6.0 to pH 8.0, more typicallypH 6.5 to 7.5), contains water as the predominant solvent, and exists ata temperature above 0° C. and below 50° C. Osmolarity is within therange that is supportive of cell viability and proliferation.

The formation of an immune complex can be detected through conventionalmethods known to the skilled artisan, for instance immunohistochemistry,immunoprecipitation, flow cytometry, immunofluorescence microscopy,ELISA, immunoblotting (for example, Western blot), magnetic resonanceimaging, CT scans, X-ray and affinity chromatography. Immunologicalbinding properties of selected antibodies may be quantified usingmethods well known in the art.

Conjugate: A complex of two molecules linked together, for example,linked together by a covalent bond. In one embodiment, an antibody islinked to an effector molecule; for example, an antibody thatspecifically binds to IL-7Rα covalently linked to an effector molecule.The linkage can be by chemical or recombinant means. In one embodiment,the linkage is chemical, wherein a reaction between the antibody moietyand the effector molecule has produced a covalent bond formed betweenthe two molecules to form one molecule. A peptide linker (short peptidesequence) can optionally be included between the antibody and theeffector molecule. Because conjugates can be prepared from two moleculeswith separate functionalities, such as an antibody and an effectormolecule, they are also sometimes referred to as “chimeric molecules.”

Conservative variants: “Conservative” amino acid substitutions are thosesubstitutions that do not substantially affect or decrease a function ofa protein, such as the ability of the protein to interact with a targetprotein. For example, an IL-7Rα-specific antibody can include up to 1,2, 3, 4, 5, 6, 7, 8, 9, or up to 10 conservative substitutions comparedto a reference antibody sequence and retain specific binding activityfor IL-7Rα. The term conservative variation also includes the use of asubstituted amino acid in place of an unsubstituted parent amino acid.

Furthermore, one of ordinary skill will recognize that individualsubstitutions, deletions or additions which alter, add or delete asingle amino acid or a small percentage of amino acids (for instanceless than 5%, in some embodiments less than 1%) in an encoded sequenceare conservative variations where the alterations result in thesubstitution of an amino acid with a chemically similar amino acid.Conservative amino acid substitution tables providing functionallysimilar amino acids are well known to one of ordinary skill in the art.The following six groups are examples of amino acids that are consideredto be conservative substitutions for one another:

1) Alanine (A), Serine (S), Threonine (T);

2) Aspartic acid (D), Glutamic acid (E);

3) Asparagine (N), Glutamine (Q);

4) Arginine (R), Lysine (K);

5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); and

6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W).

Non-conservative substitutions are those that reduce an activity orfunction of the IL-7Rα-specific antibody, such as the ability tospecifically bind to IL-7Rα. For instance, if an amino acid residue isessential for a function of the protein, even an otherwise conservativesubstitution may disrupt that activity. Thus, a conservativesubstitution does not alter the basic function of a protein of interest.

Contacting: Placement in direct physical association; includes both insolid and liquid form, which can take place either in vivo or in vitro.Contacting includes contact between one molecule and another molecule,for example the amino acid on the surface of one polypeptide, such as anantigen, that contacts another polypeptide, such as an antibody.Contacting can also include contacting a cell for example by placing anantibody in direct physical association with a cell.

Control: A reference standard. In some embodiments, the control is anegative control, such as sample obtained from a healthy patient thatdoes not have ALL. In other embodiments, the control is a positivecontrol, such as a tissue sample obtained from a patient diagnosed withALL. In still other embodiments, the control is a historical control orstandard reference value or range of values (such as a previously testedcontrol sample, such as a group of ALL patients with known prognosis oroutcome, or group of samples that represent baseline or normal values).

A difference between a test sample and a control can be an increase orconversely a decrease. The difference can be a qualitative difference ora quantitative difference, for example a statistically significantdifference. In some examples, a difference is an increase or decrease,relative to a control, of at least about 5%, such as at least about 10%,at least about 20%, at least about 30%, at least about 40%, at leastabout 50%, at least about 60%, at least about 70%, at least about 80%,at least about 90%, at least about 100%, at least about 150%, at leastabout 200%, at least about 250%, at least about 300%, at least about350%, at least about 400%, or at least about 500%.

CXCR4: C-X-C chemokine receptor type 4 (CXCR4), also known as fusin orcluster of differentiation 184 (CD184). CXCR4 is a seven transmembraneG-protein coupled receptor belonging to Class I GPCR or rhodopsin-likeGPCR family. Stromal-derived-factor-1 (SDF-1) is known to be a CXCR4ligand, and SDF-1 binding to CXCR4 is believed to promote hematopoieticstem cell homing to bone marrow. An exemplary CXCR4 protein sequence isprovided as GenBank Accession No. CAA12166.1, which is incorporated byreference herein in its entirety.

CXCR4 antagonist: An agent that decreases CXCR4 signaling activity incells. Non-limiting examples of CXCR4 antagonists include small moleculeinhibitors that of CXCR4 signaling and antibodies that specifically theextracellular region of CXCR4 on the cell surface and inhibit CXCR4signaling. A specific example of a CXCR4 antagonists includes AMD3100(Plerixafor, Genzyme Corp.). Additional CXCR4 antagonists are described,for example, in Debnath et al., Theranostics, 3(1):47-75, 2013 andGrande et al., Curr Pharm Des., 14:385-404, 2008, each of which isincorporated by reference herein in its entirety.

Degenerate variant: In the context of the present disclosure, a“degenerate variant” refers to a polynucleotide encoding a protein (forexample, an antibody that specifically binds IL-7Rα) that includes asequence that is degenerate as a result of the genetic code. There aretwenty natural amino acids, most of which are specified by more than onecodon. Therefore, all degenerate nucleotide sequences are included aslong as the amino acid sequence of the antibody that binds IL-7Rαencoded by the nucleotide sequence is unchanged.

Detectable marker: A detectable molecule (also known as a label) that isconjugated directly or indirectly to a second molecule, such as anantibody, to facilitate detection of the second molecule. For example,the detectable marker can be capable of detection by ELISA,spectrophotometry, flow cytometry, microscopy or diagnostic imagingtechniques (such as CT scans, MRIs, ultrasound, fiberoptic examination,and laparoscopic examination). Specific, non-limiting examples ofdetectable markers include fluorophores, chemiluminescent agents,enzymatic linkages, radioactive isotopes and heavy metals or compounds(for example super paramagnetic iron oxide nanocrystals for detection byMRI). In one example, a “labeled antibody” refers to incorporation ofanother molecule in the antibody. For example, the label is a detectablemarker, such as the incorporation of a radiolabeled amino acid orattachment to a polypeptide of biotinyl moieties that can be detected bymarked avidin (for example, streptavidin containing a fluorescent markeror enzymatic activity that can be detected by optical or colorimetricmethods). Various methods of labeling polypeptides and glycoproteins areknown in the art and may be used. Examples of labels for polypeptidesinclude, but are not limited to, the following: radioisotopes orradionuclides (such as ³⁵S or ¹³¹I) fluorescent labels (such asfluorescein isothiocyanate (FITC), rhodamine, lanthanide phosphors),enzymatic labels (such as horseradish peroxidase, beta-galactosidase,luciferase, alkaline phosphatase), chemiluminescent markers, biotinylgroups, predetermined polypeptide epitopes recognized by a secondaryreporter (such as a leucine zipper pair sequences, binding sites forsecondary antibodies, metal binding domains, epitope tags), or magneticagents, such as gadolinium chelates. In some embodiments, labels areattached by spacer arms of various lengths to reduce potential sterichindrance. Methods for using detectable markers and guidance in thechoice of detectable markers appropriate for various purposes arediscussed for example in Sambrook et al. (Molecular Cloning: ALaboratory Manual, 4^(th) ed, Cold Spring Harbor, New York, 2012) andAusubel et al. (In Current Protocols in Molecular Biology, John Wiley &Sons, New York, through supplement 104, 2013).

Detecting: To identify the existence, presence, or fact of something.General methods of detecting are known to the skilled artisan and may besupplemented with the protocols and reagents disclosed herein. Forexample, included herein are methods of detecting a cell that expressesIL-7Rα.

Diagnostic: Identifying the presence or nature of a pathologiccondition, such as, but not limited to, cancer. Diagnostic methodsdiffer in their sensitivity and specificity. The “sensitivity” of adiagnostic assay is the percentage of diseased individuals who testpositive (percent of true positives). The “specificity” of a diagnosticassay is one minus the false positive rate, where the false positiverate is defined as the proportion of those without the disease who testpositive. While a particular diagnostic method may not provide adefinitive diagnosis of a condition, it suffices if the method providesa positive indication that aids in diagnosis. “Prognostic” is theprobability of development (e.g., severity) of a pathologic condition,such as cancer or metastasis.

Drug: Any compound used to treat, ameliorate or prevent a disease orcondition in a subject. In some embodiments herein, the drug is achemotherapeutic agent, for example a cytotoxic agent, such as ananti-mitotic or anti-microtubule agent.

Effector molecule: A molecule intended to have or produce a desiredeffect; for example, a desired effect on a cell to which the effectormolecule is targeted. Effector molecules can include, for example,polypeptides and small molecules. In one non-limiting example, theeffector molecule is a chemotherapeutic agent. The skilled artisan willunderstand that some effector molecules may have or produce more thanone desired effect. In one example, an effector molecule is the portionof a chimeric molecule, for example a chimeric molecule that includes adisclosed antibody or fragment thereof, that is intended to have adesired effect on a cell or tissue to which the chimeric molecule istargeted.

Epitope: An antigenic determinant. These are particular chemical groupsor peptide sequences on a molecule that are antigenic, i.e. that elicita specific immune response. An antibody specifically binds a particularantigenic epitope on a polypeptide. In some examples a disclosedantibody specifically binds to an epitope on IL-7Rα.

Expression: Transcription or translation of a nucleic acid sequence. Forexample, a gene can be expressed when its DNA is transcribed into an RNAor RNA fragment, which in some examples is processed to become mRNA. Agene may also be expressed when its mRNA is translated into an aminoacid sequence, such as a protein or a protein fragment. In a particularexample, a heterologous gene is expressed when it is transcribed into anRNA. In another example, a heterologous gene is expressed when its RNAis translated into an amino acid sequence. Regulation of expression caninclude controls on transcription, translation, RNA transport andprocessing, degradation of intermediary molecules such as mRNA, orthrough activation, inactivation, compartmentalization or degradation ofspecific protein molecules after they are produced.

Expression Control Sequences: Nucleic acid sequences that regulate theexpression of a heterologous nucleic acid sequence to which it isoperatively linked. Expression control sequences are operatively linkedto a nucleic acid sequence when the expression control sequences controland regulate the transcription and, as appropriate, translation of thenucleic acid sequence. Thus expression control sequences can includeappropriate promoters, enhancers, transcription terminators, a startcodon (ATG) in front of a protein-encoding gene, splicing signal forintrons, maintenance of the correct reading frame of that gene to permitproper translation of mRNA, and stop codons. The term “controlsequences” is intended to include, at a minimum, components whosepresence can influence expression, and can also include additionalcomponents whose presence is advantageous, for example, leader sequencesand fusion partner sequences. Expression control sequences can include apromoter.

A promoter is a minimal sequence sufficient to direct transcription.Also included are those promoter elements which are sufficient to renderpromoter-dependent gene expression controllable for cell-type specific,tissue-specific, or inducible by external signals or agents; suchelements may be located in the 5′ or 3′ regions of the gene. Bothconstitutive and inducible promoters are included (see for example,Bitter et al., Methods in Enzymology 153:516-544, 1987). For example,when cloning in bacterial systems, inducible promoters such as pL ofbacteriophage lambda, plac, ptrp, ptac (ptrp-lac hybrid promoter) andthe like may be used. In one embodiment, when cloning in mammalian cellsystems, promoters derived from the genome of mammalian cells (such asmetallothionein promoter) or from mammalian viruses (such as theretrovirus long terminal repeat; the adenovirus late promoter; thevaccinia virus 7.5K promoter) can be used. Promoters produced byrecombinant DNA or synthetic techniques may also be used to provide fortranscription of the nucleic acid sequences.

A polynucleotide can be inserted into an expression vector that containsa promoter sequence, which facilitates the efficient transcription ofthe inserted genetic sequence of the host. The expression vectortypically contains an origin of replication, a promoter, as well asspecific nucleic acid sequences that allow phenotypic selection of thetransformed cells.

Expression vector: A vector comprising a recombinant polynucleotidecomprising expression control sequences operatively linked to anucleotide sequence to be expressed. An expression vector comprisessufficient cis-acting elements for expression; other elements forexpression can be supplied by the host cell or in an in vitro expressionsystem. Expression vectors include all those known in the art, such ascosmids, plasmids (e.g., naked or contained in liposomes) and viruses(e.g., lentiviruses, retroviruses, adenoviruses, and adeno-associatedviruses) that incorporate the recombinant polynucleotide.

Fc polypeptide: The polypeptide including the constant region of anantibody excluding the first constant region immunoglobulin domain. Fcregion generally refers to the last two constant region immunoglobulindomains of IgA, IgD, and IgG, and the last three constant regionimmunoglobulin domains of IgE and IgM. An Fc region may also includepart or all of the flexible hinge N-terminal to these domains. For IgAand IgM, an Fc region may or may not include the tailpiece, and may ormay not be bound by the J chain. For IgG, the Fc region includesimmunoglobulin domains Cgamma2 and Cgamma3 (Cγ2 and Cγ3) and the lowerpart of the hinge between Cgamma1 (Cγ1) and Cγ2. Although the boundariesof the Fc region may vary, the human IgG heavy chain Fc region isusually defined to include residues C226 or P230 to itscarboxyl-terminus, wherein the numbering is according to the EU index asin Kabat. For IgA, the Fc region includes immunoglobulin domains Calpha2and Calpha3 (Cα2 and Cα3) and the lower part of the hinge betweenCalpha1 (Cα1) and Cα2.

IgA: A polypeptide belonging to the class of antibodies that aresubstantially encoded by a recognized immunoglobulin alpha gene. Inhumans, this class or isotype comprises IgA₁ and IgA₂. IgA antibodiescan exist as monomers, polymers (referred to as pIgA) of predominantlydimeric form, and secretory IgA. The constant chain of wild-type IgAcontains an 18-amino-acid extension at its C-terminus called the tailpiece (tp). Polymeric IgA is secreted by plasma cells with a 15-kDapeptide called the J chain linking two monomers of IgA through theconserved cysteine residue in the tail piece.

IgG: A polypeptide belonging to the class or isotype of antibodies thatare substantially encoded by a recognized immunoglobulin gamma gene. Inhumans, this class comprises IgG₁, IgG₂, IgG₃, and IgG₄. In mice, thisclass comprises IgG₁, IgG_(2a), IgG_(2b), IgG₃.

Interleukin-7 (IL-7): A product of stromal cells and a ligand for theIL-7 receptor. IL-7 is normally required for T and B cell developmentand for survival of mature T cells. Binding of IL-7 to its receptoractivates the Jak/Stat signaling pathway, leading to cell survival andproliferation.

Interleukin-7 receptor α chain (IL-7Rα): Also known as CD127, IL-7Rα isa type-I cytokine receptor predominantly expressed by lymphocytes and itbinds to IL-7 and γc to transduce a signal that increases lymphocytesurvival and proliferation. IL-7Rα includes an extracellular domain, atrans-membrane domain, and an intracellular domain. IL-7 binding toIL-7Rα leads to heterodimerization with the common γc chain (on Tcells). On B cells, IL-7Rα heterodimerizes with TSLP and TSLP receptor.Heterodimerization triggers phosphorlyation events that ultimately leadto Stat5b homodimerization and translocation to the nucleus, whereStat5b serves as a transcription factor that induces genes involved incell survival and proliferation. Gain-of-function mutations in IL-7Rαare known to cause aberrant IL-7Rα homodimerization and phosphorylationof Stat5b, resulting in lymphocyte overproduction. An exemplary aminoacid sequence for IL-7Rα is provided as NCBI Ref. NP_002176.2, which isincorporated by reference herein as present in the database on Sep. 30,2015.

IL-7Rα-positive cancer: An abnormal (for example, malignant) growth oftissue or cells that express IL-7Rα protein, the abnormal growth oftissue or cells resulting from excessive cell division. In someembodiments, the IL-7Rα-positive cancer comprises cells thatover-express IL-7Rα. In additional embodiments, the IL-7Rα positivecancer comprises cells that express an IL-7Rα protein with one or moremutations that result in increased IL-7Rα signaling activity, forexample, the increased IL-7Rα signaling activity can result in increasedphosphorylation of Stat5b compared to a normal control. In someembodiments, the IL-7Rα-positive cancer can be a hematological cancer ora lymphoid cancer. In some embodiments, the IL-7Rα-positive cancer canbe a leukemia that expresses IL-7Rα, such as an acute lymphoblasticleukemia (for example a T-ALL or B-ALL) that expresses IL-7Rα.

Standard methods can be used to determine an expression or signalingactivity level of IL-7Rα in a cancer tissue or cell. For example, asample of the cancer tissue or cells can be taken from a subject andtested for binding to an IL-7Rα-specific antibody to determine IL-7Rαexpression, for example, to identify the cancer as having overexpressionof IL-7Rα-positive compared to a normal control. In some embodiments, asample of the cancer can be taken from a subject and tested for Stat5bphosphorylation compared to a normal control to assess IL-7Rα signalingactivity.

In some embodiments the IL-7Rα-positive cancer can be a hematological orlymphoid cancer; non-limiting examples include leukemias, for exampleacute leukemias (such as acute lymphoblastic leukemia, acute myelocyticleukemia, acute myelogenous leukemia and myeloblastic, promyelocytic,myelomonocytic, monocytic and erythroleukemia), chronic leukemias (suchas chronic myelocytic (granulocytic) leukemia, chronic myelogenousleukemia, and chronic lymphocytic leukemia), a polycythemia vera, alymphoma, Hodgkin's disease, non-Hodgkin's lymphoma (indolent and highgrade forms), multiple myeloma, Waldenstrom's macroglobulinemia, heavychain disease, myelodysplastic syndrome, hairy cell leukemia andmyelodysplasia. In several embodiments, the IL-7Rα-positive cancer canbe an acute lymphoblastic leukemia, such as T-ALL or B-ALL.

In some embodiments, the IL-7Rα-positive cancer can be a solid cancer;non-limiting examples include sarcomas and carcinomas, includingfibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenicsarcoma, and other sarcomas, synovioma, mesothelioma, Ewing's tumor,leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, lymphoid malignancy,pancreatic cancer, breast cancer, lung cancers, ovarian cancer, prostatecancer, hepatocellular carcinoma, squamous cell carcinoma, basal cellcarcinoma, adenocarcinoma, sweat gland carcinoma, medullary thyroidcarcinoma, papillary thyroid carcinoma, pheochromocytomas sebaceousgland carcinoma, papillary carcinoma, papillary adenocarcinomas,medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma,hepatoma, bile duct carcinoma, choriocarcinoma, Wilms' tumor, cervicalcancer, testicular tumor, seminoma, bladder carcinoma, and CNS tumors(such as a glioma, astrocytoma, medulloblastoma, craniopharyogioma,ependymoma, pinealoma, hemangioblastoma, acoustic neuroma,oligodendroglioma, menangioma, melanoma, neuroblastoma andretinoblastoma).

Isolated: A biological component (such as a nucleic acid, peptide,protein or protein complex, for example an antibody) that has beensubstantially separated, produced apart from, or purified away fromother biological components in the cell of the organism in which thecomponent naturally occurs, that is, other chromosomal andextra-chromosomal DNA and RNA, and proteins. Thus, isolated nucleicacids, peptides and proteins include nucleic acids and proteins purifiedby standard purification methods. The term also embraces nucleic acids,peptides and proteins prepared by recombinant expression in a host cell,as well as, chemically synthesized nucleic acids. A isolated nucleicacid, peptide or protein, for example an antibody, can be at least 50%,at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99% pure.

Linker: A bi-functional molecule that can be used to link two moleculesinto one contiguous molecule, for example, to link an effector moleculeto an antibody. In some embodiments, the provided conjugates include alinker between the effector molecule or detectable marker and anantibody. In some cases, a linker is a peptide within an antigen bindingfragment (such as an Fv fragment) which serves to indirectly bond theV_(H) and V_(L). Non-limiting examples of peptide linkers include aglycine-serine linkers such as GGGGS (SEQ ID NO: 47), GGGGSGGGGS SEQ IDNO: 48), or a GGGGSGGGGSGGGGS (SEQ ID NO: 49) linker.

The terms “conjugating,” “joining,” “bonding,” or “linking” can refer tomaking two molecules into one contiguous molecule; for example, linkingtwo polypeptides into one contiguous polypeptide, or covalentlyattaching an effector molecule or detectable marker radionuclide orother molecule to a polypeptide, such as an scFv. In the specificcontext, the terms include reference to joining a ligand, such as anantibody moiety, to an effector molecule. The linkage can be either bychemical or recombinant means. “Chemical means” refers to a reactionbetween the antibody moiety and the effector molecule such that there isa covalent bond formed between the two molecules to form one molecule.

Nucleic acid: A polymer composed of nucleotide units (ribonucleotides,deoxyribonucleotides, related naturally occurring structural variants,and synthetic non-naturally occurring analogs thereof) linked viaphosphodiester bonds, related naturally occurring structural variants,and synthetic non-naturally occurring analogs thereof. Thus, the termincludes nucleotide polymers in which the nucleotides and the linkagesbetween them include non-naturally occurring synthetic analogs, such as,for example and without limitation, phosphorothioates, phosphoramidates,methyl phosphonates, chiral-methyl phosphonates, 2-O-methylribonucleotides, peptide-nucleic acids (PNAs), and the like. Suchpolynucleotides can be synthesized, for example, using an automated DNAsynthesizer. The term “oligonucleotide” typically refers to shortpolynucleotides, generally no greater than about 50 nucleotides. It willbe understood that when a nucleotide sequence is represented by a DNAsequence (i.e., A, T, G, C), this also includes an RNA sequence (i.e.,A, U, G, C) in which “U” replaces “T. ”

Conventional notation is used herein to describe nucleotide sequences:the left-hand end of a single-stranded nucleotide sequence is the5′-end; the left-hand direction of a double-stranded nucleotide sequenceis referred to as the 5′-direction. The direction of 5′ to 3′ additionof nucleotides to nascent RNA transcripts is referred to as thetranscription direction. The DNA strand having the same sequence as anmRNA is referred to as the “coding strand;” sequences on the DNA strandhaving the same sequence as an mRNA transcribed from that DNA and whichare located 5′ to the 5′-end of the RNA transcript are referred to as“upstream sequences;” sequences on the DNA strand having the samesequence as the RNA and which are 3′ to the 3′ end of the coding RNAtranscript are referred to as “downstream sequences.”

“cDNA” refers to a DNA that is complementary or identical to an mRNA, ineither single stranded or double stranded form.

“Encoding” refers to the inherent property of specific sequences ofnucleotides in a polynucleotide, such as a gene, a cDNA, or an mRNA, toserve as templates for synthesis of other polymers and macromolecules inbiological processes having either a defined sequence of nucleotides(i.e., rRNA, tRNA and mRNA) or a defined sequence of amino acids and thebiological properties resulting therefrom. Thus, a gene encodes aprotein if transcription and translation of mRNA produced by that geneproduces the protein in a cell or other biological system. Both thecoding strand, the nucleotide sequence of which is identical to the mRNAsequence and is usually provided in sequence listings, and non-codingstrand, used as the template for transcription, of a gene or cDNA can bereferred to as encoding the protein or other product of that gene orcDNA. Unless otherwise specified, a “nucleotide sequence encoding anamino acid sequence” includes all nucleotide sequences that aredegenerate versions of each other and that encode the same amino acidsequence. Nucleotide sequences that encode proteins and RNA may includeintrons.

The nucleotides can be ribonucleotides, deoxyribonucleotides, ormodified forms of either nucleotide. The term includes single- anddouble-stranded forms of DNA.

Operably linked: A first nucleic acid sequence is operably linked with asecond nucleic acid sequence when the first nucleic acid sequence isplaced in a functional relationship with the second nucleic acidsequence. For instance, a promoter, such as the CMV promoter, isoperably linked to a coding sequence if the promoter affects thetranscription or expression of the coding sequence. Generally, operablylinked DNA sequences are contiguous and, where necessary to join twoprotein-coding regions, in the same reading frame.

Pharmaceutically acceptable carriers: The pharmaceutically acceptablecarriers of use are conventional. Remington's Pharmaceutical Sciences,by E. W. Martin, Mack Publishing Co., Easton, Pa., 19th Edition, 1995,describes compositions and formulations suitable for pharmaceuticaldelivery of the disclosed agents.

In general, the nature of the carrier will depend on the particular modeof administration being employed. For instance, parenteral formulationsusually include injectable fluids that include pharmaceutically andphysiologically acceptable fluids such as water, physiological saline,balanced salt solutions, aqueous dextrose, glycerol or the like as avehicle. For solid compositions (e.g., powder, pill, tablet, or capsuleforms), conventional non-toxic solid carriers can include, for example,pharmaceutical grades of mannitol, lactose, starch, or magnesiumstearate. In addition to biologically neutral carriers, pharmaceuticalcompositions to be administered can contain minor amounts of non-toxicauxiliary substances, such as wetting or emulsifying agents, addedpreservatives (such as on-natural preservatives), and pH bufferingagents and the like, for example sodium acetate or sorbitan monolaurate.In particular examples, the pharmaceutically acceptable carrier issterile and suitable for parenteral administration to a subject forexample, by injection. In some embodiments, the active agent andpharmaceutically acceptable carrier are provided in a unit dosage formsuch as a pill or in a selected quantity in a vial. Unit dosage formscan include one dosage or multiple dosages (for example, in a vial fromwhich metered dosages of the agents can selectively be dispensed).

Polypeptide: A polymer in which the monomers are amino acid residuesthat are joined together through amide bonds. When the amino acids arealpha-amino acids, either the L-optical isomer or the D-optical isomercan be used, the L-isomers being preferred. The terms “polypeptide” or“protein” as used herein are intended to encompass any amino acidsequence and include modified sequences such as glycoproteins. Apolypeptide includes both naturally occurring proteins, as well as thosethat are recombinantly or synthetically produced. A polypeptide has anamino terminal (N-terminal) end and a carboxy-terminal end. In someembodiments, the polypeptide is a disclosed antibody or a fragmentthereof.

Polypeptide modifications: polypeptides can be modified by a variety ofchemical techniques to produce derivatives having essentially the sameactivity and conformation as the unmodified peptides, and optionallyhaving other desirable properties. For example, carboxylic acid groupsof the protein, whether carboxyl-terminal or side chain, may be providedin the form of a salt of a pharmaceutically-acceptable cation oresterified to form a C₁-C₁₆ ester, or converted to an amide of formulaNR₁R₂ wherein R₁ and R₂ are each independently H or C₁-C₁₆ alkyl, orcombined to form a heterocyclic ring, such as a 5- or 6-membered ringAmino groups of the peptide, whether amino-terminal or side chain, maybe in the form of a pharmaceutically-acceptable acid addition salt, suchas the HCl, HBr, acetic, benzoic, toluene sulfonic, maleic, tartaric andother organic salts, or may be modified to C₁-C₁₆ alkyl or dialkyl aminoor further converted to an amide.

Hydroxyl groups of the peptide side chains can be converted to C₁-C₁₆alkoxy or to a C₁-C₁₆ ester using well-recognized techniques. Phenyl andphenolic rings of the peptide side chains can be substituted with one ormore halogen atoms, such as F, Cl, Br or I, or with C₁-C₁₆ alkyl, C₁-C₁₆alkoxy, carboxylic acids and esters thereof, or amides of suchcarboxylic acids. Methylene groups of the peptide side chains can beextended to homologous C₂-C₄ alkylenes. Thiols can be protected with anyone of a number of well-recognized protecting groups, such as acetamidegroups.

Recombinant: A recombinant nucleic acid is one that has a sequence thatis not naturally occurring or has a sequence that is made by anartificial combination of two otherwise separated segments of sequence.This artificial combination can be accomplished by chemical synthesisor, more commonly, by the artificial manipulation of isolated segmentsof nucleic acids, for example, by genetic engineering techniques. Arecombinant protein is one that has a sequence that is not naturallyoccurring or has a sequence that is made by an artificial combination oftwo otherwise separated segments of sequence. In several embodiments, arecombinant protein is encoded by a heterologous (for example,recombinant) nucleic acid that has been introduced into a host cell,such as a bacterial or eukaryotic cell. The nucleic acid can beintroduced, for example, on an expression vector having signals capableof expressing the protein encoded by the introduced nucleic acid or thenucleic acid can be integrated into the host cell chromosome.

Sequence identity: The similarity between amino acid sequences isexpressed in terms of the similarity between the sequences, otherwisereferred to as sequence identity. Sequence identity is frequentlymeasured in terms of percentage identity (or similarity or homology);the higher the percentage, the more similar the two sequences are.Homologs or variants of a polypeptide will possess a relatively highdegree of sequence identity when aligned using standard methods.

Methods of alignment of sequences for comparison are well known in theart. Various programs and alignment algorithms are described in: Smithand Waterman, Adv. Appl. Math. 2:482, 1981; Needleman and Wunsch, J.Mol. Biol. 48:443, 1970; Pearson and Lipman, Proc. Natl. Acad. Sci.U.S.A. 85:2444, 1988; Higgins and Sharp, Gene 73:237, 1988; Higgins andSharp, CABIOS 5:151, 1989; Corpet et al., Nucleic Acids Research16:10881, 1988; and Pearson and Lipman, Proc. Natl. Acad. Sci. U.S.A.85:2444, 1988. Altschul et al., Nature Genet. 6:119, 1994, presents adetailed consideration of sequence alignment methods and homologycalculations.

The NCBI Basic Local Alignment Search Tool (BLAST) (Altschul et al., J.Mol. Biol. 215:403, 1990) is available from several sources, includingthe National Center for Biotechnology Information (NCBI, Bethesda, Md.)and on the internet, for use in connection with the sequence analysisprograms blastp, blastn, blastx, tblastn and tblastx. A description ofhow to determine sequence identity using this program is available onthe NCBI website on the internet.

Homologs and variants of a V_(L) or a V_(H) of an antibody thatspecifically binds a polypeptide are typically characterized bypossession of at least about 75%, for example at least about 80%, 85%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identitycounted over the full length alignment with the amino acid sequence ofinterest. Proteins with even greater similarity to the referencesequences will show increasing percentage identities when assessed bythis method, such as at least 80%, at least 85%, at least 90%, at least95%, at least 98%, or at least 99% sequence identity. When less than theentire sequence is being compared for sequence identity, homologs andvariants will typically possess at least 80% sequence identity overshort windows of 10-20 amino acids, and may possess sequence identitiesof at least 85% or at least 90% or 95% depending on their similarity tothe reference sequence. Methods for determining sequence identity oversuch short windows are available at the NCBI website on the internet.One of skill in the art will appreciate that these sequence identityranges are provided for guidance only; it is entirely possible thatstrongly significant homologs could be obtained that fall outside of theranges provided.

Terms used to describe sequence relationships between two or morenucleotide sequences or amino acid sequences include “referencesequence,” “selected from,” “comparison window,” “identical,”“percentage of sequence identity,” “substantially identical,”“complementary,” and “substantially complementary.”

For sequence comparison of nucleic acid sequences, typically onesequence acts as a reference sequence, to which test sequences arecompared. When using a sequence comparison algorithm, test and referencesequences are entered into a computer, subsequence coordinates aredesignated, if necessary, and sequence algorithm program parameters aredesignated. Default program parameters are used. Methods of alignment ofsequences for comparison are well known in the art. Optimal alignment ofsequences for comparison can be conducted, e.g., by the local homologyalgorithm of Smith & Waterman, Adv. Appl. Math. 2:482, 1981, by thehomology alignment algorithm of Needleman & Wunsch, J. Mol. Biol.48:443, 1970, by the search for similarity method of Pearson & Lipman,Proc. Nat'l. Acad. Sci. USA 85:2444, 1988, by computerizedimplementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA inthe Wisconsin Genetics Software Package, Genetics Computer Group, 575Science Dr., Madison, Wis.), or by manual alignment and visualinspection (see, e.g., Sambrook et al. (Molecular Cloning: A LaboratoryManual, 4^(th) ed, Cold Spring Harbor, New York, 2012) and Ausubel etal. (In Current Protocols in Molecular Biology, John Wiley & Sons, NewYork, through supplement 104, 2013). One example of a useful algorithmis PILEUP. PILEUP uses a simplification of the progressive alignmentmethod of Feng & Doolittle, J. Mol. Evol. 35:351-360, 1987. The methodused is similar to the method described by Higgins & Sharp, CABIOS5:151-153, 1989. Using PILEUP, a reference sequence is compared to othertest sequences to determine the percent sequence identity relationshipusing the following parameters: default gap weight (3.00), default gaplength weight (0.10), and weighted end gaps. PILEUP can be obtained fromthe GCG sequence analysis software package, e.g., version 7.0 (Devereauxet al., Nuc. Acids Res. 12:387-395, 1984.

Another example of algorithms that are suitable for determining percentsequence identity and sequence similarity are the BLAST and the BLAST2.0 algorithm, which are described in Altschul et al., J. Mol. Biol.215:403-410, 1990 and Altschul et al., Nucleic Acids Res. 25:3389-3402,1977. Software for performing BLAST analyses is publicly availablethrough the National Center for Biotechnology Information(ncbi.nlm.nih.gov). The BLASTN program (for nucleotide sequences) usesas defaults a word length (W) of 11, alignments (B) of 50, expectation(E) of 10, M=5, N=-4, and a comparison of both strands. The BLASTPprogram (for amino acid sequences) uses as defaults a word length (W) of3, and expectation (E) of 10, and the BLOSUM62 scoring matrix (seeHenikoff & Henikoff, Proc. Natl. Acad. Sci. USA 89:10915, 1989). Anoligonucleotide is a linear polynucleotide sequence of up to about 100nucleotide bases in length.

Specifically bind: When referring to an antibody or antigen bindingfragment, refers to a binding reaction which determines the presence ofa target protein, peptide, or polysaccharide in the presence of aheterogeneous population of proteins and other biologics. Thus, underdesignated conditions, an antibody binds preferentially to a particulartarget protein, peptide or polysaccharide (such as IL-7Rα) and does notbind in a significant amount to other proteins or polysaccharidespresent in the sample or subject. Specific binding can be determined bymethods known in the art. With reference to an antibody-antigen complex,specific binding of the antigen and antibody has a K_(D) of less thanabout 10⁻⁷ Molar, such as less than about 10⁻⁸ Molar, 10⁻⁹, or even lessthan about 10⁻¹⁰ Molar.

K_(D) refers to the dissociation constant for a given interaction, suchas a polypeptide ligand interaction or an antibody antigen interaction.For example, for the bimolecular interaction of an antibody or antigenbinding fragment and an antigen it is the concentration of theindividual components of the bimolecular interaction divided by theconcentration of the complex.

The antibodies disclosed herein specifically bind to a defined target(or multiple targets, in the case of a bispecific antibody). Thus, anantibody that specifically binds to an epitope on IL-7Rα is an antibodythat binds substantially to IL-7Rα, including cells or tissue expressingIL-7Rα, substrate to which the IL-7Rα is attached, or IL-7Rα in abiological specimen. It is, of course, recognized that a certain degreeof non-specific interaction may occur between an antibody or conjugateincluding an antibody (such as an antibody that specifically bindsIL-7Rα or conjugate including such antibody) and a non-target (such as acell that does not express IL-7Rα). Typically, specific binding resultsin a much stronger association between the antibody and protein or cellsbearing the antigen than between the antibody and protein or cellslacking the antigen. Specific binding typically results in greater than2-fold, such as greater than 5-fold, greater than 10-fold, or greaterthan 100-fold increase in amount of bound antibody (per unit time) to aprotein including the epitope or cell or tissue expressing the targetepitope as compared to a protein or cell or tissue lacking this epitope.Specific binding to a protein under such conditions requires an antibodythat is selected for its specificity for a particular protein. A varietyof immunoassay formats are appropriate for selecting antibodies or otherligands specifically immunoreactive with a particular protein. Forexample, solid-phase ELISA immunoassays are routinely used to selectmonoclonal antibodies specifically immunoreactive with a protein. SeeHarlow & Lane, Antibodies, A Laboratory Manual, 2^(nd) ed., Cold SpringHarbor Publications, New York (2013), for a description of immunoassayformats and conditions that can be used to determine specificimmunoreactivity.

Subject: Living multi-cellular vertebrate organisms, a category thatincludes human and non-human mammals. In an example, a subject is ahuman. In a particular example, the subject is a pediatric subject, suchas a human child age 2-5 years old. In an additional example, a subjectis selected that has ALL (such as T-ALL) or is at risk of having ALL(such as T-ALL).

Therapeutically effective amount: The amount of an agent (such as anIL-7Rα specific antibody or a conjugate including an IL-7Rα specificantibody) that alone, or together with one or more additional agents,induces the desired response, such as, for example treatment of anIL-7Rα-positive cancer, in a subject. When administered to a subject, adosage will generally be used that will achieve target tissueconcentrations that has been shown to achieve a desired in vitro effect.Ideally, a therapeutically effective amount provides a therapeuticeffect without causing a substantial cytotoxic effect in the subject.

In one example, a desired response is to decrease the size, volume, ornumber (such as metastases) of IL-7Rα-positive cancer cells in asubject, and/or neoplastic lesions or number of leukemia cells in bloodin a subject. For example, the agent or agents can decrease the size,volume, or number of IL-7Rα-positive cancer cells, and/or neoplasticlesions or number of leukemia cells in blood by a desired amount, forexample by at least 5%, at least 10%, at least 15%, at least 20%, atleast 25%, at least 30%, at least 50%, at least 75%, at least 90%, or atleast 95% as compared to a response in the absence of the agent.

Several preparations disclosed herein are administered intherapeutically effective amounts. A therapeutically effective amount ofan antibody that specifically binds IL-7Rα or antigen binding fragmentthereof, or conjugate thereof (or a composition including one or more ofthese molecules) that is administered to a human or veterinary subjectwill vary depending upon a number of factors associated with thatsubject, for example the overall health of the subject. Atherapeutically effective amount can be determined by varying the dosageand measuring the resulting therapeutic response, such as the regressionof an IL-7Rα-positive cancer. Therapeutically effective amounts also canbe determined through various in vitro, in vivo or in situ immunoassays.The disclosed agents can be administered in a single dose, or in severaldoses, as needed to obtain the desired response. However, thetherapeutically effective amount of can be dependent on the sourceapplied, the subject being treated, the severity and type of thecondition being treated, and the manner of administration.

A therapeutically effective amount encompasses a fractional dose thatcontributes in combination with previous or subsequent administrationsto attaining a therapeutic response. For example, a therapeuticallyeffective amount of an agent can be administered in a single dose, or inseveral doses, for example daily, during a course of treatment lastingseveral days or weeks. However, the therapeutically effective amount candepend on the subject being treated, the severity and type of thecondition being treated, and the manner of administration. A unit dosageform of the agent can be packaged in a therapeutic amount, or inmultiples of the therapeutic amount, for example, in a vial (e.g., witha pierceable lid) or syringe having sterile components.

Toxin: An effector molecule that induces cytotoxicity when it contacts acell. Specific, non-limiting examples of toxins include, but are notlimited to, abrin, ricin, auristatins (such as monomethyl auristatin E(MMAE; see for example, Francisco et al., Blood, 102: 1458-1465, 2003))and monomethyl auristatin F (MMAF; see, for example, Doronina et al.,BioConjugate Chem., 17: 114-124, 2006), maytansinoids (such as DM1; see,for example, Phillips et al., Cancer Res., 68:9280-9290, 2008),Pseudomonas exotoxin (PE, such as PE35, PE37, PE38, and PE40),diphtheria toxin (DT), botulinum toxin, saporin, restrictocin orgelonin, or modified toxins thereof, or other toxic agents that directlyor indirectly inhibit cell growth or kill cells. For example, PE and DTare highly toxic compounds that typically bring about death throughliver toxicity. PE and DT, however, can be modified into a form for useas an immunotoxin by removing the native targeting component of thetoxin (such as the domain Ia of PE and the B chain of DT) and replacingit with a different targeting moiety, such as an antibody.

Transformed: A transformed cell is a cell into which a nucleic acidmolecule has been introduced by molecular biology techniques. As usedherein, the term transformation encompasses all techniques by which anucleic acid molecule might be introduced into such a cell, includingtransfection with viral vectors, transformation with plasmid vectors,and introduction of DNA by electroporation, lipofection, and particlegun acceleration.

Treating or Preventing a disease: “Preventing” a disease refers toinhibiting the full development of a disease. “Treating” refers to atherapeutic intervention that ameliorates a sign or symptom of a diseaseor pathological condition after it has begun to develop, such as areduction in tumor burden or a decrease in the number of size ofmetastases. “Ameliorating” refers to the reduction in the number orseverity of signs or symptoms of a disease, such as cancer.

Vector: A nucleic acid molecule as introduced into a host cell, therebyproducing a transformed host cell. Recombinant DNA vectors are vectorshaving recombinant DNA. A vector can include nucleic acid sequences thatpermit it to replicate in a host cell, such as an origin of replication.A vector can also include one or more selectable marker genes and othergenetic elements known in the art. Viral vectors are recombinant nucleicacid vectors having at least some nucleic acid sequences derived fromone or more viruses. A replication deficient viral vector is a vectorthat requires complementation of one or more regions of the viral genomerequired for replication due to a deficiency in at least onereplication-essential gene function. For example, such that the viralvector does not replicate in typical host cells, especially those in ahuman patient that could be infected by the viral vector in the courseof a therapeutic method.

III. DESCRIPTION OF SEVERAL EMBODIMENTS A. Monoclonal Antibodies andAntigen Binding Fragments

Isolated monoclonal antibodies and antigen binding fragments thatspecifically bind an epitope on the extracellular domain of IL-7Rα areprovided. In several embodiments, the antibodies or antigen bindingfragments can specifically bind to IL-7Rα on the surface of a cell, suchas a T-ALL cell.

This disclosure provides the novel 4A10 and 2B8 antibodies and variantsthereof, including antigen binding fragments. Epitope mapping andcompetition binding studies show that the disclosed antibodies andantigen binding fragments specifically bind to non-overlapping epitopeson the extracellular domain of IL-7Rα.

The disclosed antibodies and antigen binding fragments are surprisinglyeffective for treatment of ALL, such as T-ALL and B-ALL. For example, asdiscussed in Example 1, a chimeric antibody including the 4A10 heavy andlight chain variable regions and human IgG1 constant regions prolongedsurvival and mediated ADCC killing of IL-7Rα-positive cancer cells in anin vivo model of T-ALL.

In some embodiments, the antibodies and antigen binding fragmentsinclude a V_(H) and a V_(L) and specifically bind to IL-7Rα. In someembodiments, the antibody or antigen binding fragment includes a V_(H)comprising one or more (i.e., one, two or all three) HCDRs from the 4A10or 2B8 antibody and specifically binds to the extracellular domain ofIL-7Rα. In some embodiments, the antibody or antigen binding fragmentincludes a V_(L) comprising one or more (i.e., one, two or all three)LCDRs from one of the 4A10 or 2B8 antibody and specifically binds to theextracellular domain of IL-7Rα. In several embodiments, the antibody orantigen binding fragment includes a V_(H) and a V_(L) including theHCDR1, HCDR2, and HCDR3, and LCDR1, LCDR2, and LCDR3, respectively, ofthe 4A10 or 2B8 antibody, and specifically binds to the extracellulardomain of IL-7Rα.

In some embodiments, the antibody or antigen binding fragment includes aHCDR1, a HCDR2, a HCDR3, a LCDR1, a LCDR2, and a LCDR3 comprising aminoacid sequences that are at least 90%, for example, at least 91%, atleast 92%, at least 93%, at least 94%, at least 95%, at least 96%, atleast 97%, at least 98%, at least 99% or 100% identical to the aminoacid sequences of the CDRs of one of the 4A10 or 2B8 antibody, whereinthe antibody specifically binds to the extracellular domain of IL-7Rα.

The person of ordinary skill in the art will understand that various CDRnumbering schemes (such as the Kabat, Chothia or IMGT numbering schemes)can be used to determine CDR positions. The amino acid sequence and theCDR positions of the heavy and light chain of the 4A10 and 2B8antibodies according to the Kabat numbering scheme are shown in Table 1.The discussion of monoclonal antibodies below refers to monoclonalantibodies that include a V_(H) and a V_(L) including CDRs withreference to the Kabat numbering scheme (unless the context indicatesotherwise). In some embodiments, the antibody or antigen bindingfragment includes one or more Kabat CDRs, such as those listed in Table1.

TABLE 1 Kabat CDR sequences of IL-7Rα specific antibodies. CDR SEQ ID NO4A10 V_(H) SEQ ID NO: 1 V_(H) positions CDR protein sequence HCDR1 31-35SYWMH  5 HCDR2 50-66 EIDPSDSYTNDNQKFKG  6 HCDR3  99-111 RLYSNSYYYAMDY  74A10 V_(L) SEQ ID NO: 2 V_(L) positions A.A. Sequence LCDR1 24-34KASQDIKKYIA  8 LCDR2 50-56 YTSTLQP  9 LCDR3 89-96 LQYDNLLT 10 2B8 V_(H)SEQ ID NO: 3 V_(H) positions CDR protein sequence HCDR1 31-35 DYYMH 11HCDR2 50-66 YIYPDNGGNGYNQKFKG 12 HCDR3  99-109 GTYYDGSYFDY 13 2B8 V_(L)SEQ ID NO: 4 V_(L) positions A.A. Sequence LCDR1 24-34 KASQDVSTTVA 14LCDR2 50-56 SASYRYT 15 LCDR3 89-97 QQHYSIPRT 16

4A10

In some embodiments, the antibody or antigen binding fragment can bebased on or derived from the 4A10 antibody. For example, in someembodiments, the antibody or antigen binding fragment includes a V_(H)including a HCDR1, a HCDR2, and/or a HCDR3 including amino acids 31-35,50-66, and 99-111 of SEQ ID NO: 1, respectively, wherein the antibody orantigen binding fragment specifically binds to the extracellular domainof IL-7Rα. In further embodiments, the antibody or antigen bindingfragment includes a V_(L) including a LCDR1, a LCDR2, and/or a LCDR3including amino acids 24-34, 50-56, and 89-96 of SEQ ID NO: 2,respectively, wherein the antibody or antigen binding fragmentspecifically binds to the extracellular domain of IL-7Rα. In additionalembodiments, the antibody or antigen binding fragment includes a V_(H)including a HCDR1, a HCDR2, and/or a HCDR3 including amino acids 31-35,50-66, and 99-111 of SEQ ID NO: 1, respectively, and a V_(L) including aLCDR1, a LCDR2, and/or a LCDR3 including amino acids 24-34, 50-56, and89-96 of SEQ ID NO: 2, respectively, wherein the antibody or antigenbinding fragment specifically binds to the extracellular domain ofIL-7Rα.

In some embodiments, the antibody or antigen binding fragment includes aV_(H) including a HCDR1, a HCDR2, and a HCDR3 including amino acids31-35, 50-66, and 99-111 of SEQ ID NO: 1, respectively, wherein theantibody or antigen binding fragment specifically binds to theextracellular domain of IL-7Rα. In further embodiments, the antibody orantigen binding fragment includes a V_(L) including a LCDR1, a LCDR2,and a LCDR3 including amino acids 24-34, 50-56, and 89-96 of SEQ ID NO:2, respectively, wherein the antibody or antigen binding fragmentspecifically binds to the extracellular domain of IL-7Rα. In additionalembodiments, the antibody or antigen binding fragment includes a V_(H)including a HCDR1, a HCDR2, and a HCDR3 including amino acids 31-35,50-66, and 99-111 of SEQ ID NO: 1, respectively, and a V_(L) including aLCDR1, a LCDR2, and a LCDR3 including amino acids 24-34, 50-56, and89-96 of SEQ ID NO: 2, respectively, wherein the antibody or antigenbinding fragment specifically binds to the extracellular domain ofIL-7Rα.

In some embodiments, an isolated antibody or antigen binding fragmentincludes at least one CDR with a sequence that has at least 95% sequenceidentity to any one of SEQ ID NOs: 5-10, wherein the antibodyspecifically binds to IL-7Rα. In some embodiments, the antibody orantigen binding fragment includes a V_(H) including a HCDR1, a HCDR2,and a HCDR3 including amino acid sequences at least 90% (such as atleast 95%, at least 96%, at least 97%, at least 98%, or at least 99%)identical to amino acids 31-35, 50-66, and 99-111, respectively, of SEQID NO: 1, wherein the antibody or antigen binding fragment specificallybinds to the extracellular domain of IL-7Rα. In some embodiments, theantibody or antigen binding fragment includes a V_(L) including a LCDR1,a LCDR2, and a LCDR3 including amino acid sequences at least 90% (suchas at least 95%, at least 96%, at least 97%, at least 98%, or at least99%) identical to amino acids amino acids 24-34, 50-56, and 89-96,respectively, of SEQ ID NO: 2, wherein the antibody or antigen bindingfragment specifically binds to the extracellular domain of IL-7Rα. Inadditional embodiments, the antibody or antigen binding fragmentincludes a V_(H) including a HCDR1, a HCDR2, and a HCDR3 including aminoacid sequences at least 90% (such as at least 95%, at least 96%, atleast 97%, at least 98%, or at least 99%) identical to amino acids31-35, 50-66, and 99-111, respectively, of SEQ ID NO: 1, and a V_(L)including a LCDR1, a LCDR2, and a LCDR3 including amino acid sequencesat least 90% (such as at least 95%, at least 96%, at least 97%, at least98%, or at least 99%) identical to amino acids amino acids 24-34, 50-56,and 89-96, respectively, of SEQ ID NO: 2, wherein the antibody orantigen binding fragment specifically binds to the extracellular domainof IL-7Rα.

In some embodiments, the antibody or antigen binding fragment includes aV_(H) including an amino acid sequence at least 90% (such as at least95%, at least 96%, at least 97%, at least 98%, or at least 99%) to theamino acid sequence set forth as SEQ ID NO: 1, wherein the antibody orantigen binding fragment specifically binds to the extracellular domainof IL-7Rα. In more embodiments, the antibody or antigen binding fragmentincludes a V_(L) including an amino acid sequence at least 90% (such asat least 95%, at least 96%, at least 97%, at least 98%, or at least 99%)to the amino acid sequence set forth as SEQ ID NO: 2, wherein theantibody or antigen binding fragment specifically binds to theextracellular domain of IL-7Rα. In additional embodiments, the antibodyor antigen binding fragment includes a V_(H) including an amino acidsequence at least 90% (such as at least 95%, at least 96%, at least 97%,at least 98%, or at least 99%) to the amino acid sequence set forth asSEQ ID NO: 1, and a V_(L) including an amino acid sequence at least 90%(such as at least 95%, at least 96%, at least 97%, at least 98%, or atleast 99%) to the amino acid sequence set forth as SEQ ID NO: 2, whereinthe antibody or antigen binding fragment specifically binds to theextracellular domain of IL-7Rα.

In additional embodiments, the antibody or antigen binding fragmentincludes a V_(H) including the amino acid sequence set forth as one ofSEQ ID NO: 1, wherein the antibody or antigen binding fragmentspecifically binds to the extracellular domain of IL-7Rα. In moreembodiments, the antibody or antigen binding fragment includes a V_(L)including the amino acid sequence set forth as SEQ ID NO: 2, wherein theantibody or antigen binding fragment specifically binds to theextracellular domain of IL-7Rα. In some embodiments, the antibody orantigen binding fragment includes a V_(H) and a V_(L) including theamino acid sequences set forth as SEQ ID NOs: 1 and 2, respectively,wherein the antibody or antigen binding fragment specifically binds tothe extracellular domain of IL-7Rα.

In some embodiments, the antibody or antigen binding fragment includes aHCDR1, a HCDR2, and a HCDR3, comprising the amino acid sequences setforth as SEQ ID NOs: 5, 6, and 7, respectively, wherein the antibody orantigen binding fragment specifically binds to the extracellular domainof IL-7Rα. In some embodiments, the antibody or antigen binding fragmentincludes a LCDR1, a LCDR2, and a LCDR3, comprising the amino acidsequences set forth as SEQ ID NOs: 8, 9, and 10, respectively, whereinthe antibody or antigen binding fragment specifically binds to theextracellular domain of IL-7Rα. In some embodiments, the antibody orantigen binding fragment includes a HCDR1, a HCDR2, a HCDR3, a LCDR1, aLCDR2, and a LCDR3, comprising the amino acid sequences set forth as SEQID NOs: 5, 6, 7, 8, 9, and 10, respectively, wherein the antibody orantigen binding fragment specifically binds to the extracellular domainof IL-7Rα.

2B8

In some embodiments, the antibody or antigen binding fragment can bebased on or derived from the 4A10 antibody. For example, in someembodiments, the antibody or antigen binding fragment includes a V_(H)including a HCDR1, a HCDR2, and/or a HCDR3 including amino acids 31-35,50-66, and 99-109 of SEQ ID NO: 3, respectively, wherein the antibody orantigen binding fragment specifically binds to the extracellular domainof IL-7Rα. In further embodiments, the antibody or antigen bindingfragment includes a V_(L) including a LCDR1, a LCDR2, and/or a LCDR3including amino acids 24-34, 50-56, and 89-97 of SEQ ID NO: 4,respectively, wherein the antibody or antigen binding fragmentspecifically binds to the extracellular domain of IL-7Rα. In additionalembodiments, the antibody or antigen binding fragment includes a V_(H)including a HCDR1, a HCDR2, and/or a HCDR3 including amino acids 31-35,50-66, and 99-109 of SEQ ID NO: 3, respectively, and a V_(L) including aLCDR1, a LCDR2, and/or a LCDR3 including amino acids 24-34, 50-56, and89-97 of SEQ ID NO: 4, respectively, wherein the antibody or antigenbinding fragment specifically binds to the extracellular domain ofIL-7Rα.

In some embodiments, the antibody or antigen binding fragment includes aV_(H) including a HCDR1, a HCDR2, and a HCDR3 including amino acids31-35, 50-66, and 99-109 of SEQ ID NO: 3, respectively, wherein theantibody or antigen binding fragment specifically binds to theextracellular domain of IL-7Rα. In further embodiments, the antibody orantigen binding fragment includes a V_(L) including a LCDR1, a LCDR2,and a LCDR3 including amino acids 24-34, 50-56, and 89-97 of SEQ ID NO:4, respectively, wherein the antibody or antigen binding fragmentspecifically binds to the extracellular domain of IL-7Rα. In additionalembodiments, the antibody or antigen binding fragment includes a V_(H)including a HCDR1, a HCDR2, and a HCDR3 including amino acids 31-35,50-66, and 99-109 of SEQ ID NO: 3, respectively, and a V_(L) including aLCDR1, a LCDR2, and a LCDR3 including amino acids 24-34, 50-56, and89-97 of SEQ ID NO: 4, respectively, wherein the antibody or antigenbinding fragment specifically binds to the extracellular domain ofIL-7Rα.

In some embodiments, an isolated antibody or antigen binding fragmentincludes at least one CDR with a sequence that has at least 95% sequenceidentity to any one of SEQ ID NOs: 11-16, wherein the antibodyspecifically binds to IL-7Rα. In some embodiments, the antibody orantigen binding fragment includes a V_(H) including a HCDR1, a HCDR2,and a HCDR3 including amino acid sequences at least 90% (such as atleast 95%, at least 96%, at least 97%, at least 98%, or at least 99%)identical to amino acids 31-35, 50-66, and 99-109, respectively, of SEQID NO: 3, wherein the antibody or antigen binding fragment specificallybinds to the extracellular domain of IL-7Rα. In some embodiments, theantibody or antigen binding fragment includes a V_(L) including a LCDR1,a LCDR2, and a LCDR3 including amino acid sequences at least 90% (suchas at least 95%, 96%, 97%, 98%, or 99%) identical to amino acids aminoacids 24-34, 50-56, and 89-97, respectively, of SEQ ID NO: 4, whereinthe antibody or antigen binding fragment specifically binds to theextracellular domain of IL-7Rα. In additional embodiments, the antibodyor antigen binding fragment includes a V_(H) including a HCDR1, a HCDR2,and a HCDR3 including amino acid sequences at least 90% (such as atleast 95%, at least 96%, at least 97%, at least 98%, or at least 99%)identical to amino acids 31-35, 50-66, and 99-109, respectively, of SEQID NO: 3, and a V_(L) including a LCDR1, a LCDR2, and a LCDR3 includingamino acid sequences at least 90% (such as at least 95%, at least 96%,at least 97%, at least 98%, or at least 99%) identical to amino acidsamino acids 24-34, 50-56, and 89-97, respectively, of SEQ ID NO: 4,wherein the antibody or antigen binding fragment specifically binds tothe extracellular domain of IL-7Rα.

In some embodiments, the antibody or antigen binding fragment includes aV_(H) including an amino acid sequence at least 90% (such as at least95%, at least 96%, at least 97%, at least 98%, or at least 99%) to theamino acid sequence set forth as SEQ ID NO: 3, wherein the antibody orantigen binding fragment specifically binds to the extracellular domainof IL-7Rα. In more embodiments, the antibody or antigen binding fragmentincludes a V_(L) including an amino acid sequence at least 90% (such asat least 95%, at least 96%, at least 97%, at least 98%, or at least 99%)identical to the amino acid sequence set forth as SEQ ID NO: 4, whereinthe antibody or antigen binding fragment specifically binds to theextracellular domain of IL-7Rα. In additional embodiments, the antibodyor antigen binding fragment includes a V_(H) including an amino acidsequence at least 90% (such as at least 95%, at least 96%, at least 97%,at least 98%, or at least 99%) identical to the amino acid sequence setforth as SEQ ID NO: 3, and a V_(L) including an amino acid sequence atleast 90% (such as at least 95%, at least 96%, at least 97%, at least98%, or at least 99%) identical to the amino acid sequence set forth asSEQ ID NO: 4, wherein the antibody or antigen binding fragmentspecifically binds to the extracellular domain of IL-7Rα.

In additional embodiments, the antibody or antigen binding fragmentincludes a V_(H) including the amino acid sequence set forth as one ofSEQ ID NO: 3, wherein the antibody or antigen binding fragmentspecifically binds to the extracellular domain of IL-7Rα. In moreembodiments, the antibody or antigen binding fragment includes a V_(L)including the amino acid sequence set forth as SEQ ID NO: 4, wherein theantibody or antigen binding fragment specifically binds to theextracellular domain of IL-7Rα. In some embodiments, the antibody orantigen binding fragment includes a V_(H) and a V_(L) including theamino acid sequences set forth as SEQ ID NOs: 3 and 4, respectively,wherein the antibody or antigen binding fragment specifically binds tothe extracellular domain of IL-7Rα.

In some embodiments, the antibody or antigen binding fragment includes aHCDR1, a HCDR2, and a HCDR3, comprising the amino acid sequences setforth as SEQ ID NOs: 11, 12, and 13, respectively, wherein the antibodyor antigen binding fragment specifically binds to the extracellulardomain of IL-7Rα. In some embodiments, the antibody or antigen bindingfragment includes a LCDR1, a LCDR2, and a LCDR3, comprising the aminoacid sequences set forth as SEQ ID NOs: 14, 15, and 16, respectively,wherein the antibody or antigen binding fragment specifically binds tothe extracellular domain of IL-7Rα. In some embodiments, the antibody orantigen binding fragment includes a HCDR1, a HCDR2, a HCDR3, a LCDR1, aLCDR2, and a LCDR3, comprising the amino acid sequences set forth as SEQID NOs: 11, 12, 13, 14, 15, and 16, respectively, wherein the antibodyor antigen binding fragment specifically binds to the extracellulardomain of IL-7Rα.

1. Additional Description of Antibodies and Antigen Binding Fragments

The 4A10 and 2B8 antibodies were originally isolated from mousehybridoma cell lines. In some embodiments, a humanized antibody isprovided that includes the CDRs of the 4A10 or 2B8 antibody and humanframework regions. Chimeric antibodies (for example, including mousevariable regions and human constant regions) are also provided. Theantibody or antigen binding fragment can include any suitable frameworkregion, such as (but not limited to) a human framework region. Humanframework regions, and mutations that can be made in a human antibodyframework regions, are known in the art (see, for example, in U.S. Pat.No. 5,585,089, which is incorporated herein by reference).Alternatively, a heterologous framework region, such as, but not limitedto a mouse or monkey framework region, can be included in the heavy orlight chain of the antibodies. (See, for example, Jones et al., Nature321:522, 1986; Riechmann et al., Nature 332:323, 1988; Verhoeyen et al.,Science 239:1534, 1988; Carter et al., Proc. Natl. Acad. Sci. U.S.A.89:4285, 1992; Sandhu, Crit. Rev. Biotech.12:437, 1992; and Singer etal., J. Immunol.150:2844, 1993.)

The antibody can be of any isotype. The antibody can be, for example, anIgM or an IgG antibody, such as IgG1, IgG₂, IgG₃, or IgG₄. The class ofan antibody that specifically binds IL-7Rα can be switched with another.In one aspect, a nucleic acid molecule encoding V_(L) or V_(H) isisolated using methods well-known in the art, such that it does notinclude any nucleic acid sequences encoding the constant region of thelight or heavy chain, respectively. In a non-limiting example, the V_(H)amino acid sequence is set forth as SEQ ID NO: 1, and the V_(L) aminoacid sequence is set forth as SEQ ID NO: 2. In another non-limitingexample, the V_(H) amino acid sequence is set forth as SEQ ID NO: 3, andthe V_(L) amino acid sequence is set forth as SEQ ID NO: 4. A nucleicacid molecule encoding V_(L) or V_(H) is then operatively linked to anucleic acid sequence encoding a C_(L) or C_(H) from a different classof immunoglobulin molecule. This can be achieved using a vector ornucleic acid molecule that comprises a C_(L) or C_(H) chain, as known inthe art. For example, an antibody that specifically binds IL-7Rα, thatwas originally IgG may be class switched to an IgM. Class switching canbe used to convert one IgG subclass to another, such as from IgG₁ toIgG₂, IgG₃, or IgG₄.

In some examples, the disclosed antibodies are oligomers of antibodies,such as dimers, trimers, tetramers, pentamers, hexamers, septamers,octomers and so on.

In several embodiments, the disclosed antibody can mediate ADCC killingof IL-7Rα positive cells, such as T-ALL cells. In some embodiments, thedisclosed antibody can inhibit IL-7 induced signaling through the IL-7Rin cells, such as T-ALL cells. Methods of evaluating IL-7-inducedsignaling through the IL-7® are known and include, for example,evaluation of pSTAT5 induction in the presence of IL-7.

(a) Binding Affinity

In several embodiments, the antibody or antigen binding fragment canspecifically bind IL-7Rα with an affinity (e.g., measured by K_(D)) ofno more than 1.0×10⁻⁸M, no more than 5.0×10⁻⁸M, no more than 1.0×10⁻⁹M,no more than 5.0×10⁻⁹M, no more than 1.0×10⁻¹⁰M, no more than5.0×10⁻¹⁰M, or no more than 1.0×10⁻¹¹M. K_(D) can be measured, forexample, by a radiolabeled antigen binding assay (RIA) performed withthe Fab version of an antibody of interest and its antigen using, knownmethods. In one assay, solution binding affinity of Fabs for antigen ismeasured by equilibrating Fab with a minimal concentration of(¹²⁵I)-labeled antigen in the presence of a titration series ofunlabeled antigen, then capturing bound antigen with an anti-Fabantibody-coated plate (see, e.g., Chen et al., J. Mol. Biol. 293:865-881(1999)). To establish conditions for the assay, MICROTITER® multi-wellplates (Thermo Scientific) are coated overnight with 5 μg/ml of acapturing anti-Fab antibody (Cappel Labs) in 50 mM sodium carbonate (pH9.6), and subsequently blocked with 2% (w/v) bovine serum albumin in PBSfor two to five hours at room temperature (approximately 23° C.). In anon-adsorbent plate (Nunc #269620), 100 μM or 26 μM [¹²⁵I]-antigen aremixed with serial dilutions of a Fab of interest (e.g., consistent withassessment of the anti-VEGF antibody, Fab-12, in Presta et al., CancerRes. 57:4593-4599 (1997)). The Fab of interest is then incubatedovernight; however, the incubation may continue for a longer period(e.g., about 65 hours) to ensure that equilibrium is reached.Thereafter, the mixtures are transferred to the capture plate forincubation at room temperature (e.g., for one hour). The solution isthen removed and the plate washed eight times with 0.1% polysorbate 20(TWEEN-20®) in PBS. When the plates have dried. 150 μ/well ofscintillant (MICROSCINT-20™; Packard) is added, and the plates arecounted on a TOPCOUNT™ gamma counter (Packard) for ten minutes.Concentrations of each Fab that give less than or equal to 20% ofmaximal binding are chosen for use in competitive binding assays.

In another assay, K_(D) can be measured using surface plasmon resonanceassays using a Biacore™ T-100 or a Biacore™-3000 instrument (GE LifeSciences, Inc., Piscataway, N.J.) at 25° C. with immobilized antigen CM5chips at ˜10 response units (RU). Briefly, carboxymethylated dextranbiosensor chips (CMS, GE Life Sciences, Inc.) are activated withN-ethyl-N′-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC) andN-hydroxysuccinimide (NHS) according to the supplier's instructions.Antigen is diluted with 10 mM sodium acetate, pH 4.8, to 5 μg/ml (˜0.2μM) before injection at a flow rate of 5 μl/min to achieve approximately10-50 response units (RU's) of coupled protein. Following the injectionof antigen, 1 M ethanolamine is injected to block unreacted aminogroups. For kinetics measurements, two-fold serial dilutions of Fab(0.78 nM to 500 nM) are injected in PBS with 0.05% polysorhate 20(TWEEN-20™) surfactant (PBST) at 25° C. at a flow rate of approximately25 μl/min. Association rates (k_(on)) and dissociation rates (k_(off))are calculated using a simple one-to-one Langmuir binding model(Biacore™ Evaluation Software version 3.2) by simultaneously fitting theassociation and dissociation phases of the sensorgrams. The equilibriumdissociation constant (K_(d)) is calculated as the ratio k_(off)/k_(on).See, e.g., Chen et al., J. Mol. Biol. 293:865-881 (1999). If the on-rateexceeds 10⁶ M⁻¹s⁻ by the surface plasmon resonance assay above, then theon-rate can be determined by using a fluorescent quenching techniquethat measures the increase or decrease in fluorescence emissionintensity (excitation=295 nm; emission=340 nm, 16 nm band-pass) at 25°C. of a 20 nM anti-antigen antibody (Fab form) in PBS, pH 7.2, in thepresence of increasing concentrations of antigen as measured in aspectrometer, such as a stop-flow equipped spectrophometer (AvivInstruments) or a 8000-series SLIM-AMINCO™ spectrophotometer(ThermoSpectronic) with a stifled cuvette.

(b) Multispecific Antibodies

In some embodiments, the antibody or antigen binding fragment isincluded on a multispecific antibody, such as a bi-specific antibody.Such multispecific antibodies can be produced by known methods, such ascrosslinking two or more antibodies, antigen binding fragments (such asscFvs) of the same type or of different types. Exemplary methods ofmaking multispecific antibodies include those described in PCT Pub. No.WO2013/163427, which is incorporated by reference herein in itsentirety. Suitable crosslinkers include those that areheterobifunctional, having two distinctly reactive groups separated byan appropriate spacer (such as m-maleimidobenzoyl-N-hydroxysuccinimideester) or homobifunctional (such as disuccinimidyl suberate). Suchlinkers are available from Pierce Chemical Company, Rockford, Ill.

In some embodiments, the antibody or antigen binding fragment isincluded on a bispecific antibody that that specifically binds to IL-7Rαand further specifically binds to CD3, such as in the context of aB-specific T cell engager (BiTE). Examples of CD3 binding domains thatcan be included on the bispecific antibody or antigen binding fragmentare known and include those disclosed in PCT Pub. No. WO2013/163427,which is incorporated by reference herein in its entirety.

Various types of multi-specific antibodies are known. Bispecific singlechain antibodies can be encoded by a single nucleic acid molecule.Examples of bispecific single chain antibodies, as well as methods ofconstructing such antibodies are known in the art (see, e.g., U.S. Pat.Nos. 8,076,459, 8,017,748, 8,007,796, 7,919,089, 7,820,166, 7,635,472,7,575,923, 7,435,549, 7,332,168, 7,323,440, 7,235,641, 7,229,760,7,112,324, 6,723,538, incorporated by reference herein). Additionalexamples of bispecific single chain antibodies can be found in PCTapplication No. WO 99/54440; Mack, J. Immunol., 158:3965-3970, 1997;Mack, PNAS, 92:7021-7025, 1995; Kufer, Cancer Immunol. Immunother.,45:193-197, 1997; Loffler, Blood, 95:2098-2103, 2000; and Bruhl, J.Immunol., 166:2420-2426, 2001. Production of bispecific Fab-scFv(“bibody”) molecules are described, for example, in Schoonjans et al.(J. Immunol. 165:7050-57, 2000) and Willems et al. (J Chromatogr BAnalyt Technol Biomed Life Sci. 786:161-76, 2003). For bibodies, a scFvmolecule can be fused to one of the VL-CL (L) or VH-CH1 chains, e.g., toproduce a bibody one scFv is fused to the C-term of a Fab chain.

(c) Fragments

Antigen binding fragments are encompassed by the present disclosure,such as Fab, F(ab′)₂, and Fv which include a V_(H) and V_(L) (forexample, with the CDRs of the 4A10 or 2B8 mAb) and specifically bindIL-7Rα. These antibody fragments retain the ability to selectively bindwith the antigen and are “antigen-binding” fragments. Non-limitingexamples of such fragments include:

(1) Fab, the fragment which contains a monovalent antigen-bindingfragment of an antibody molecule, can be produced by digestion of wholeantibody with the enzyme papain to yield an intact light chain and aportion of one heavy chain;

(2) Fab′, the fragment of an antibody molecule can be obtained bytreating whole antibody with pepsin, followed by reduction, to yield anintact light chain and a portion of the heavy chain; two Fab′ fragmentsare obtained per antibody molecule;

(3) (Fab′)₂, the fragment of the antibody that can be obtained bytreating whole antibody with the enzyme pepsin without subsequentreduction; F(ab′)₂ is a dimer of two Fab′ fragments held together by twodisulfide bonds;

(4) Fv, a genetically engineered fragment containing the V_(L) and V_(L)expressed as two chains; and

(⁵) Single chain antibody (such as scFv), defined as a geneticallyengineered molecule containing the V_(H) and the V_(L) linked by asuitable polypeptide linker as a genetically fused single chain molecule(see, e.g., Ahmad et al., Clin. Dev. Immunol., 2012,doi:10.1155/2012/980250; Marbry, IDrugs, 13:543-549, 2010). Theintramolecular orientation of the V_(H)-domain and the V_(L)-domain in ascFv, is not decisive for the provided antibodies (e.g., for theprovided multispecific antibodies). Thus, scFvs with both possiblearrangements (V_(H)-domain-linker domain-V_(L)-domain;V_(L)-domain-linker domain-V_(H)-domain) may be used.

(6) A dimer of a single chain antibody (scFV₂), defined as a dimer of ascFV. This has also been termed a “miniantibody.”

Methods of making these fragments are known in the art (see for example,Harlow and Lane, Antibodies: A Laboratory Manual, 2^(nd), Cold SpringHarbor Laboratory, New York, 2013).

In some embodiments, the antibody binding fragment can be an Fvantibody, which is typically about 25 kDa and contain a completeantigen-binding site with three CDRs per each heavy chain and each lightchain. To produce F_(v) antibodies, the V_(H) and the V_(L) can beexpressed from two individual nucleic acid constructs in a host cell. Inanother example, the V_(H) amino acid sequence of the antigen bindingfragment includes the CDRs from SEQ ID NO: 1 and/or the sequence setforth as one of SEQ ID NO: 1, and the V_(L) amino acid sequence of theantigen binding fragment includes the CDRs from SEQ ID NO: 2, and/or thesequence set forth as SEQ ID NO: 2. In another example, the V_(H) aminoacid sequence of the antigen binding fragment includes the CDRs from SEQID NO: 3 and/or the sequence set forth as one of SEQ ID NO: 3, and theV_(L) amino acid sequence of the antigen binding fragment includes theCDRs from SEQ ID NO: 4, and/or the sequence set forth as SEQ ID NO: 4.

If the V_(H) and the V_(L) are expressed non-contiguously, the chains ofthe Fv antibody are typically held together by noncovalent interactions.However, these chains tend to dissociate upon dilution, so methods havebeen developed to crosslink the chains through glutaraldehyde,intermolecular disulfides, or a peptide linker. Thus, in one example,the Fv can be a disulfide stabilized Fv (dsFv), wherein the V_(H) andthe V_(L) are chemically linked by disulfide bonds.

In an additional example, the Fv fragments include V_(H) and V_(L)chains connected by a peptide linker. These single-chain antigen bindingproteins (scFv) can be prepared by constructing a nucleic acid moleculeencoding the V_(H) and V_(L) domains connected by an oligonucleotide.The nucleic acid molecule is inserted into an expression vector, whichis subsequently introduced into a host cell such as a mammalian cell.The recombinant host cells synthesize a single polypeptide chain with alinker peptide bridging the two V domains. Methods for producing scFvsare known in the art (see Whitlow et al., Methods: a Companion toMethods in Enzymology, Vol. 2, page 97, 1991; Bird et al., Science242:423, 1988; U.S. Pat. No. 4,946,778; Pack et al., Bio/Technology11:1271, 1993; Ahmad et al., Clin. Dev. Immunol., 2012,doi:10.1155/2012/980250; Marbry, IDrugs, 13:543-549, 2010). Dimers of asingle chain antibody (scFV₂), are also contemplated.

Antigen binding fragments can be prepared by proteolytic hydrolysis ofthe antibody or by expression in a host cell (such as an E. coli cell)of DNA encoding the fragment. Antigen binding fragments can also beobtained by pepsin or papain digestion of whole antibodies byconventional methods. For example, antigen binding fragments can beproduced by enzymatic cleavage of antibodies with pepsin to provide a 5Sfragment denoted F(ab′)₂. This fragment can be further cleaved using athiol reducing agent, and optionally a blocking group for the sulfhydrylgroups resulting from cleavage of disulfide linkages, to produce 3.5SFab′ monovalent fragments. Alternatively, an enzymatic cleavage usingpepsin produces two monovalent Fab′ fragments and an Fc fragmentdirectly (see U.S. Pat. No. 4,036,945 and U.S. Pat. No. 4,331,647, andreferences contained therein; Nisonhoff et al., Arch. Biochem. Biophys.89:230, 1960; Porter, Biochem. J. 73:119, 1959; Edelman et al., Methodsin Enzymology, Vol. 1, page 422, Academic Press, 1967; and Coligan etal. at sections 2.8.1-2.8.10 and 2.10.1-2.10.4).

Other methods of cleaving antibodies, such as separation of heavy chainsto form monovalent light-heavy chain fragments, further cleavage offragments, or other enzymatic, chemical, or genetic techniques may alsobe used, so long as the fragments bind to the antigen that is recognizedby the intact antibody.

In some embodiments, one or more (such as all) of the heavy chain and/orlight chain CDRs from a disclosed antibody (such as the 4A10 or 2B8) isexpressed on the surface of another protein, such as a scaffold protein.The expression of domains of antibodies on the surface of a scaffoldingprotein are known in the art (see e.g., Liu et al., J. Virology 85(17):8467-8476, 2011). Such expression creates a chimeric protein thatretains the binding for IL-7Rα. In some specific embodiments, one ormore of the heavy chain CDRs is grafted onto a scaffold protein, such asone or more of heavy chain CDR1, CDR2, and/or CDR3. One or more CDRs canalso be included in a diabody or another type of single chain antibodymolecule.

(d) Variants

In certain embodiments, amino acid sequence variants of the antibodiesprovided herein are provided. For example, it may be desirable toimprove the binding affinity and/or other biological properties of theantibody Amino acid sequence variants of an antibody may be prepared byintroducing appropriate modifications into the nucleotide sequenceencoding the antibody, or by peptide synthesis. Such modificationsinclude, for example, deletions from, and/or insertions into and/orsubstitutions of residues within the amino acid sequences of theantibody. Any combination of deletion, insertion, and substitution canbe made to arrive at the final construct, provided that the finalconstruct possesses the desired characteristics, e.g., antigen-binding.

In certain embodiments, antibody variants having one or more amino acidsubstitutions are provided. Sites of interest for substitutionalmutagenesis include the CDRs and the framework regions. Amino acidsubstitutions may be introduced into an antibody of interest and theproducts screened for a desired activity, e.g., retained/improvedantigen binding, decreased immunogenicity, or improved ADCC or CDC.

The variants typically retain amino acid residues necessary for correctfolding and stabilizing between the V_(H) and the V_(L) regions, andwill retain the charge characteristics of the residues in order topreserve the low pI and low toxicity of the molecules. Amino acidsubstitutions can be made in the V_(H) and the V_(L) regions to increaseyield.

In some embodiments, the V_(H) and V_(L) of the antibody or antigenbinding fragment each include up to 10 (such as up to 1, up to 2, up to3, up to 4, up to 5, up to 6, up to 7, up to 8, or up to 9) amino acidsubstitutions (such as conservative amino acid substitutions) comparedto the amino acid sequences set forth as SEQ ID NOs: 1 and 2,respectively, and the antibody or antigen binding fragment maintainsspecific binding activity for IL-7Rα. In some embodiments, the V_(H) andV_(L) of the antibody or antigen binding fragment each include up to 10(such as up to 1, up to 2, up to 3, up to 4, up to 5, up to 6, up to 7,up to 8, or up to 9) amino acid substitutions (such as conservativeamino acid substitutions) compared to the amino acid sequences set forthas SEQ ID NOs: 3 and 4, respectively, and the antibody or antigenbinding fragment maintains specific binding activity for IL-7Rα.

In some embodiments, the antibody or antigen binding fragment caninclude up to 10 (such as up to 1, up to 2, up to 3, up to 4, up to 5,up to 6, up to 7, up to 8, or up to 9) amino acid substitutions (such asconservative amino acid substitutions) in the framework regions of theheavy chain of the antibody, or the light chain of the antibody, or theheavy and light chains of the antibody, compared to a known frameworkregion, or compared to the framework regions of the 410 or 2B8 antibody,and maintains the specific binding activity for IL-7Rα.

In certain embodiments, substitutions, insertions, or deletions mayoccur within one or more CDRs so long as such alterations do notsubstantially reduce the ability of the antibody to bind antigen. Forexample, conservative alterations (e.g., conservative substitutions asprovided herein) that do not substantially reduce binding affinity maybe made in CDRs. In certain embodiments of the variant V_(H) and V_(L)sequences provided above, each CDR either is unaltered, or contains nomore than one, two or three amino acid substitutions.

To increase binding affinity of the antibody, the V_(L) and V_(H)segments can be randomly mutated, such as within H-CDR3 region or theL-CDR3 region, in a process analogous to the in vivo somatic mutationprocess responsible for affinity maturation of antibodies during anatural immune response. Thus in vitro affinity maturation can beaccomplished by amplifying V_(H) and V_(L) regions using PCR primerscomplementary to the H-CDR3 or L-CDR3, respectively. In this process,the primers have been “spiked” with a random mixture of the fournucleotide bases at certain positions such that the resultant PCRproducts encode V_(H) and V_(L) segments into which random mutationshave been introduced into the V_(H) and/or V_(L) CDR3 regions. Theserandomly mutated V_(H) and V_(L) segments can be tested to determine thebinding affinity for IL-7Rα. In particular examples, the V_(H) aminoacid sequence is one of SEQ ID NOs: 1 or 3. In other examples, the V_(L)amino acid sequence is one of SEQ ID NOs: 2 or 4. Methods of in vitroaffinity maturation are known (see, e.g., Chowdhury, Methods Mol. Biol.207:179-196 (2008)), and Hoogenboom et al. in Methods in MolecularBiology 178:1-37 (O′Brien et al., ed., Human Press, Totowa, N.J.,(2001).)

A useful method for identification of residues or regions of an antibodythat may be targeted for mutagenesis is called “alanine scanningmutagenesis” as described by Cunningham and Wells (1989) Science,244:1081-1085. In this method, a residue or group of target residues(e.g., charged residues such as arg, asp, his, lys, and glu) areidentified and replaced by a neutral or negatively charged amino acid(e.g., alanine or polyalanine) to determine whether the interaction ofthe antibody with antigen is affected. Further substitutions may beintroduced at the amino acid locations demonstrating functionalsensitivity to the initial substitutions. Alternatively, oradditionally, a crystal structure of an antigen-antibody complex is usedto identify contact points between the antibody and antigen. Suchcontact residues and neighboring residues may be targeted or eliminatedas candidates for substitution. Variants may be screened to determinewhether they contain the desired properties.

In certain embodiments, an antibody or antigen binding fragment isaltered to increase or decrease the extent to which the antibody orantigen binding fragment is glycosylated. Addition or deletion ofglycosylation sites may be conveniently accomplished by altering theamino acid sequence such that one or more glycosylation sites is createdor removed.

Where the antibody comprises an Fc region, the carbohydrate attachedthereto may be altered. Native antibodies produced by mammalian cellstypically comprise a branched, biantennary oligosaccharide that isgenerally attached by an N-linkage to Asn297 of the CH₂ domain of the Fcregion. See, e.g., Wright et al. TIBTECH 15:26-32 (1997). Theoligosaccharide may include various carbohydrates, e.g., mannose,N-acetyl glucosamine (GlcNAc), galactose, and sialic acid, as well as afucose attached to a GlcNAc in the “stem” of the biantennaryoligosaccharide structure. In some embodiments, modifications of theoligosaccharide in an antibody may be made in order to create antibodyvariants with certain improved properties.

In one embodiment, antibody variants are provided having a carbohydratestructure that lacks fucose attached (directly or indirectly) to an Fcregion. For example, the amount of fucose in such antibody may be from1% to 80%, from 1% to 65%, from 5% to 65% or from 20% to 40%. The amountof fucose is determined by calculating the average amount of fucosewithin the sugar chain at Asn297, relative to the sum of allglycostructures attached to Asn 297 (e.g. complex, hybrid and highmannose structures) as measured by MALDI-TOF mass spectrometry, asdescribed in WO 2008/077546, for example. Asn297 refers to theasparagine residue located at about position 297 in the Fc region;however, Asn297 may also be located about ±3 amino acids upstream ordownstream of position 297, i.e., between positions 294 and 300, due tominor sequence variations in antibodies. Such fucosylation variants mayhave improved ADCC function. See, e.g., US Patent Publication Nos. US2003/0157108 (Presta, L.); US 2004/0093621 (Kyowa Hakko Kogyo Co., Ltd).Examples of publications related to “defucosylated” or“fucose-deficient” antibody variants include: US 2003/0157108; WO2000/61739; WO 2001/29246; US 2003/0115614; US 2002/0164328; US2004/0093621; US 2004/0132140; US 2004/0110704; US 2004/0110282; US2004/0109865; WO 2003/085119; WO 2003/084570; WO 2005/035586; WO2005/035778; WO2005/053742; WO2002/031140; Okazaki et al. J. Mol. Biol.336:1239-1249 (2004); Yamane-Ohnuki et al. Biotech. Bioeng. 87: 614(2004). Examples of cell lines capable of producing defucosylatedantibodies include Lec 13 CHO cells deficient in protein fucosylation(Ripka et al. Arch. Biochem. Biophys. 249:533-545 (1986); US Pat Appl NoUS 2003/0157108 A1, Presta, L; and WO 2004/056312 Al, Adams et al.,especially at Example 11), and knockout cell lines, such asalpha-1,6-fucosyltransferase gene, FUT8, knockout CHO cells (see, e.g.,Yamane-Ohnuki et al. Biotech. Bioeng. 87: 614 (2004); Kanda, Y. et al.,Biotechnol. Bioeng., 94(4):680-688 (2006); and WO2003/085107).

Antibodies variants are further provided with bisected oligosaccharides,e.g., in which a biantennary oligosaccharide attached to the Fc regionof the antibody is bisected by GlcNAc. Such antibody variants may havereduced fucosylation and/or improved ADCC function. Examples of suchantibody variants are described, e.g., in WO 2003/011878 (Jean-Mairet etal.); U.S. Pat. No. 6,602,684 (Umana et al.); and US 2005/0123546 (Umanaet al.). Antibody variants with at least one galactose residue in theoligosaccharide attached to the Fc region are also provided. Suchantibody variants may have improved CDC function. Such antibody variantsare described, e.g., in WO 1997/30087 (Patel et al.); WO 1998/58964(Raju, S.); and WO 1999/22764 (Raju, S.).

In several embodiments, the constant region of the antibody includes oneor more amino acid substitutions to optimize in vivo half-life of theantibody. The serum half-life of IgG Abs is regulated by the neonatal Fcreceptor (FcRn). Thus, in several embodiments, the antibody includes anamino acid substitution that increases binding to the FcRn. Several suchsubstitutions are known to the person of ordinary skill in the art, suchas substitutions at IgG constant regions T250Q and M428L (see, e.g.,Hinton et al., J Immunol., 176:346-356, 2006); M428L and N434S (the “LS”mutation, see, e.g., Zalevsky, et al., Nature Biotechnology, 28:157-159,2010); N434A (see, e.g., Petkova et al., Int. Immunol., 18:1759-1769,2006); T307A, E380A, and N434A (see, e.g., Petkova et al., Int.Immunol., 18:1759-1769, 2006); and M252Y, S254T, and T256E (see, e.g.,Dall'Acqua et al., J. Biol. Chem., 281:23514-23524, 2006).The disclosedantibodies and antigen binding fragments can be linked to a Fcpolypeptide including any of the substitutions listed above, forexample, the Fc polypeptide can be an IgG including the M428L and N434Ssubstitutions.

In some embodiments, the constant region of the antibody includes one ofmore amino acid substitutions to optimize antibody-dependentcell-mediated cytotoxicity (ADCC). ADCC is mediated primarily through aset of closely related Fc γ receptors. In some embodiments, the antibodyincludes one or more amino acid substitutions that increase binding toFc γRIIIa. Several such substitutions are known to the person ofordinary skill in the art, such as IgG substitutions at constant regionsS239D and I332E (see, e.g., Lazar et al., Proc. Natl., Acad. Sci.U.S.A., 103:4005-4010, 2006); and S239D, A330L, and I332E (see, e.g.,Lazar et al., Proc. Natl., Acad. Sci. U.S.A., 103:4005-4010, 2006).

Combinations of the above substitutions are also included, to generatean IgG constant region with increased binding to FcRn and FcyRIIIa. Thecombinations increase antibody half-life and ADCC. For example, suchcombination include antibodies with the following amino acidsubstitution in the Fc region:

(1) S239D/I332E and T250Q/M428L;

(2) S239D/I332E and M428L/N434S;

(3) S239D/I332E and N434A;

(4) S239D/I332E and T307A/E380A/N434A;

(5) S239D/I332E and M252Y/S254T/T256E;

(6) S239D/A330L/I332E and T250Q/M428L;

(7) S239D/A330L/I332E and M428L/N434S;

(8) S239D/A330L/I332E and N434A;

(9) S239D/A330L/I332E and T307A/E380A/N434A; or

(10) S239D/A330L/I332E and M252Y/S254T/T256E.

In some examples, the antibodies, or an antigen binding fragment thereofis modified such that it is directly cytotoxic to infected cells, oruses natural defenses such as complement, antibody dependent cellularcytotoxicity (ADCC), or phagocytosis by macrophages.

In certain embodiments, an antibody provided herein may be furthermodified to contain additional nonproteinaceous moieties that are knownin the art and readily available. The moieties suitable forderivatization of the antibody include but are not limited to watersoluble polymers. Non-limiting examples of water soluble polymersinclude, but are not limited to, polyethylene glycol (PEG), copolymersof ethylene glycol/propylene glycol, carboxymethylcellulose, dextran,polyvinyl alcohol, polyvinyl pyrrolidone, poly-1,3-dioxolane,poly-1,3,6-trioxane, ethylene/maleic anhydride copolymer, polyaminoacids(either homopolymers or random copolymers), and dextran or poly(n-vinylpyrrolidone)polyethylene glycol, propropylene glycol homopolymers,prolypropylene oxide/ethylene oxide co-polymers, polyoxyethylatedpolyols (e.g., glycerol), polyvinyl alcohol, and mixtures thereof.Polyethylene glycol propionaldehyde may have advantages in manufacturingdue to its stability in water. The polymer may be of any molecularweight, and may be branched or unbranched. The number of polymersattached to the antibody may vary, and if more than one polymer areattached, they can be the same or different molecules. In general, thenumber and/or type of polymers used for derivatization can be determinedbased on considerations including, but not limited to, the particularproperties or functions of the antibody to be improved, whether theantibody derivative will be used in a therapy under defined conditions,etc.

The antibody or antigen binding fragment can be derivatized or linked toanother molecule (such as another peptide or protein). In general, theantibody or antigen binding fragment is derivatized such that thebinding to IL-7Rα is not affected adversely by the derivatization orlabeling. For example, the antibody or antigen binding fragment can befunctionally linked (by chemical coupling, genetic fusion, noncovalentassociation or otherwise) to one or more other molecular entities, suchas another antibody (for example, a bi-specific antibody or a diabody),a detectable marker, an effector molecule, or a protein or peptide thatcan mediate association of the antibody or antibody portion with anothermolecule (such as a streptavidin core region or a polyhistidine tag).

B. Conjugates

The antibodies and antigen binding fragments that specifically bind toan epitope on IL-7Rα can be conjugated to an agent, such as an effectormolecule or detectable marker, using any number of means known to thoseof skill in the art. Both covalent and noncovalent attachment means maybe used. One of skill in the art will appreciate that various effectormolecules and detectable markers can be used, including (but not limitedto) toxins and radioactive agents such as ¹²⁵¹I, ³²P, ¹⁴C, ³H, and ³⁵Sand other labels, target moieties and ligands, etc.

The choice of a particular effector molecule or detectable markerdepends on the particular target molecule or cell, and the desiredbiological effect. Thus, for example, the effector molecule can be acytotoxin that is used to bring about the death of a particular targetcell (such as a T-ALL cell).

The procedure for attaching an effector molecule or detectable marker toan antibody or antigen binding fragment varies according to the chemicalstructure of the effector. Polypeptides typically contain a variety offunctional groups; such as carboxylic acid (COOH), free amine (—NH₂) orsulfhydryl (—SH) groups, which are available for reaction with asuitable functional group on a polypeptide to result in the binding ofthe effector molecule or detectable marker. Alternatively, the antibodyor antigen binding fragment is derivatized to expose or attachadditional reactive functional groups. The derivatization may involveattachment of any of a number of known linker molecules such as thoseavailable from Pierce Chemical Company, Rockford, Ill. The linker can beany molecule used to join the antibody or antigen binding fragment tothe effector molecule or detectable marker. The linker is capable offorming covalent bonds to both the antibody or antigen binding fragmentand to the effector molecule or detectable marker. Suitable linkers arewell known to those of skill in the art and include, but are not limitedto, straight or branched-chain carbon linkers, heterocyclic carbonlinkers, or peptide linkers. Where the antibody or antigen bindingfragment and the effector molecule or detectable marker arepolypeptides, the linkers may be joined to the constituent amino acidsthrough their side groups (such as through a disulfide linkage tocysteine) or to the alpha carbon amino and carboxyl groups of theterminal amino acids.

In several embodiments, the linker can include a spacer element, which,when present, increases the size of the linker such that the distancebetween the effector molecule or the detectable marker and the antibodyor antigen binding fragment is increased. Exemplary spacers are known tothe person of ordinary skill, and include those listed in U.S. Pat. Nos.7,964,5667, 498,298, 6,884,869, 6,323,315, 6,239,104, 6,034,065,5,780,588, 5,665,860, 5,663,149, 5,635,483, 5,599,902, 5,554,725,5,530,097, 5,521,284, 5,504,191, 5,410,024, 5,138,036, 5,076,973,4,986,988, 4,978,744, 4,879,278, 4,816,444, and 4,486,414, as well asU.S. Pat. Pub. Nos. 20110212088 and 20110070248, each of which isincorporated by reference in its entirety.

In some embodiments, the linker is cleavable under intracellularconditions, such that cleavage of the linker releases the effectormolecule or detectable marker from the antibody or antigen bindingfragment in the intracellular environment. In yet other embodiments, thelinker is not cleavable and the effector molecule or detectable markeris released, for example, by antibody degradation. In some embodiments,the linker is cleavable by a cleaving agent that is present in theintracellular environment (for example, within a lysosome or endosome orcaveolea). The linker can be, for example, a peptide linker that iscleaved by an intracellular peptidase or protease enzyme, including, butnot limited to, a lysosomal or endosomal protease. In some embodiments,the peptide linker is at least two amino acids long or at least threeamino acids long. However, the linker can be 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14 or 15 amino acids long, such as 1-2, 1-3, 2-5, 3-10, 3-15,1-5, 1-10, 1-15, amino acids long. Proteases can include cathepsins Band D and plasmin, all of which are known to hydrolyze dipeptide drugderivatives resulting in the release of active drug inside target cells(see, for example, Dubowchik and Walker, 1999, Pharm. Therapeutics83:67-123). For example, a peptide linker that is cleavable by thethiol-dependent protease cathepsin-B, can be used (for example, aPhenylalanine-Leucine or a Glycine-Phenylalanine-Leucine-Glycinelinker). Other examples of such linkers are described, for example, inU.S. Pat. No. 6,214,345, incorporated herein by reference. In a specificembodiment, the peptide linker cleavable by an intracellular protease isa Valine-Citruline linker or a Phenylalanine-Lysine linker (see, forexample, U.S. Pat. No. 6,214,345, which describes the synthesis ofdoxorubicin with the Valine-Citruline linker).

In other embodiments, the cleavable linker is pH-sensitive, i.e.,sensitive to hydrolysis at certain pH values. Typically, thepH-sensitive linker is hydrolyzable under acidic conditions. Forexample, an acid-labile linker that is hydrolyzable in the lysosome (forexample, a hydrazone, semicarbazone, thiosemicarbazone, cis-aconiticamide, orthoester, acetal, ketal, or the like) can be used. (See, forexample, U.S. Pat. Nos. 5,122,368; 5,824,805; 5,622,929; Dubowchik andWalker, 1999, Pharm. Therapeutics 83:67-123; Neville et al., 1989, Biol.Chem. 264:14653-14661.) Such linkers are relatively stable under neutralpH conditions, such as those in the blood, but are unstable at below pH5.5 or 5.0, the approximate pH of the lysosome. In certain embodiments,the hydrolyzable linker is a thioether linker (such as, for example, athioether attached to the therapeutic agent via an acylhydrazone bond(see, for example, U.S. Pat. No. 5,622,929).

In yet other embodiments, the linker is cleavable under reducingconditions (for example, a disulfide linker). A variety of disulfidelinkers are known in the art, including, for example, those that can beformed using SATA (N-succinimidyl-S-acetylthioacetate), SPDP(N-succinimidyl-3-(2-pyridyldithio)propionate), SPDB(N-succinimidyl-3-(2-pyridyldithio)butyrate) and SMPT(N-succinimidyl-oxycarbonyl-alpha-methyl-alpha-(2-pyridyl-dithio)toluene)-, SPDB and SMPT. (See, for example, Thorpe et al., 1987, Cancer Res.47:5924-5931; Wawrzynczak et al., In Immunoconjugates: AntibodyConjugates in Radioimagery and Therapy of Cancer (C. W. Vogel ed.,Oxford U. Press, 1987); Phillips et al., Cancer Res. 68:92809290, 2008).See also U.S. Pat. No. 4,880,935.)

In yet other specific embodiments, the linker is a malonate linker(Johnson et al., 1995, Anticancer Res. 15:1387-93), a maleimidobenzoyllinker (Lau et al., 1995, Bioorg-Med-Chem. 3(10):1299-1304), or a3′-N-amide analog (Lau et al., 1995, Bioorg-Med-Chem. 3(10):1305-12).

In several embodiments, the linker is resistant to cleavage in anextracellular environment. Whether or not a linker is resistant tocleavage in an extracellular environment can be determined, for example,by incubating the conjugate containing the linker of interest withplasma for a predetermined time period (for example, 2, 4, 8, 16, or 24hours) and then quantitating the amount of free effector molecule ordetectable marker present in the plasma. A variety of exemplary linkersthat can be used in conjugates are described in WO 2004010957, U.S.Publication No. 2006/0074008, U.S. Publication No. 20050238649, and U.S.Publication No. 2006/0024317, each of which is incorporated by referenceherein in its entirety.

In view of the large number of methods that have been reported forattaching a variety of radiodiagnostic compounds, radiotherapeuticcompounds, labels (such as enzymes or fluorescent molecules), toxins,and other agents to antibodies one skilled in the art will be able todetermine a suitable method for attaching a given agent to an antibodyor antigen binding fragment or other polypeptide. For example, theantibody or antigen binding fragment can be conjugated with effectormolecules such as small molecular weight drugs such as MonomethylAuristatin E (MMAE), Monomethyl Auristatin F (MMAF), maytansine,maytansine derivatives, including the derivative of maytansine known asDM1 (also known as mertansine), or other agents to make an antibody drugconjugate (ADC). In several embodiments, conjugates of an antibody orantigen binding fragment and one or more small molecule toxins, such asa calicheamicin, maytansinoids, dolastatins, auristatins, atrichothecene, and CC1065, and the derivatives of these toxins that havetoxin activity, are provided.

The antibody or antigen binding fragment can be conjugated with adetectable marker; for example, a detectable marker capable of detectionby ELISA, spectrophotometry, flow cytometry, microscopy or diagnosticimaging techniques (such as computed tomography (CT), computed axialtomography (CAT) scans, magnetic resonance imaging (MRI), nuclearmagnetic resonance imaging NMRI), magnetic resonance tomography (MTR),ultrasound, fiberoptic examination, and laparoscopic examination).Specific, non-limiting examples of detectable markers includefluorophores, chemiluminescent agents, enzymatic linkages, radioactiveisotopes and heavy metals or compounds (for example super paramagneticiron oxide nanocrystals for detection by MRI). For example, usefuldetectable markers include fluorescent compounds, including fluorescein,fluorescein isothiocyanate, rhodamine,5-dimethylamine-l-napthalenesulfonyl chloride, phycoerythrin, lanthanidephosphors and the like. Bioluminescent markers are also of use, such asluciferase, Green fluorescent protein (GFP), Yellow fluorescent protein(YFP). An antibody or antigen binding fragment can also be conjugatedwith enzymes that are useful for detection, such as horseradishperoxidase, β-galactosidase, luciferase, alkaline phosphatase, glucoseoxidase and the like. When an antibody or antigen binding fragment isconjugated with a detectable enzyme, it can be detected by addingadditional reagents that the enzyme uses to produce a reaction productthat can be discerned. For example, when the agent horseradishperoxidase is present the addition of hydrogen peroxide anddiaminobenzidine leads to a colored reaction product, which is visuallydetectable. An antibody or antigen binding fragment may also beconjugated with biotin, and detected through indirect measurement ofavidin or streptavidin binding. It should be noted that the avidinitself can be conjugated with an enzyme or a fluorescent label.

The antibody or antigen binding fragment can be conjugated with aparamagnetic agent, such as gadolinium. Paramagnetic agents such assuperparamagnetic iron oxide are also of use as labels. Antibodies canalso be conjugated with lanthanides (such as europium and dysprosium),and manganese. An antibody or antigen binding fragment may also belabeled with a predetermined polypeptide epitopes recognized by asecondary reporter (such as leucine zipper pair sequences, binding sitesfor secondary antibodies, metal binding domains, epitope tags).

The antibody or antigen binding fragment can also be conjugated with aradiolabeled amino acid. The radiolabel may be used for both diagnosticand therapeutic purposes. For instance, the radiolabel may be used todetect IL-7Rα and IL-7Rα expressing cells by x-ray, emission spectra, orother diagnostic techniques. Examples of labels for polypeptidesinclude, but are not limited to, the following radioisotopes orradionucleotides: ³H, ¹⁴C, ¹⁵N, ³⁵S, ⁹⁰Y, ⁹⁹Tc, ¹¹¹In, ¹²⁵I, ¹³¹I.

Means of detecting such detectable markers are well known to those ofskill in the art. Thus, for example, radiolabels may be detected usingphotographic film or scintillation counters, fluorescent markers may bedetected using a photodetector to detect emitted illumination. Enzymaticlabels are typically detected by providing the enzyme with a substrateand detecting the reaction product produced by the action of the enzymeon the substrate, and colorimetric labels are detected by simplyvisualizing the colored label.

The average number of effector molecule or detectable marker moietiesper antibody or antigen binding fragment in a conjugate can range, forexample, from 1 to 20 moieties per antibody or antigen binding fragment.In certain embodiments, the average number of effector molecule ordetectable marker moieties per antibody or antigen binding fragment in aconjugate range from about 1 to about 2, from about 1 to about 3, about1 to about 8; from about 2 to about 6; from about 3 to about 5; or fromabout 3 to about 4. The loading (for example, effector molecule/antibodyratio) of an conjugate may be controlled in different ways, for example,by: (i) limiting the molar excess of effector molecule-linkerintermediate or linker reagent relative to antibody, (ii) limiting theconjugation reaction time or temperature, (iii) partial or limitingreductive conditions for cysteine thiol modification, (iv) engineeringby recombinant techniques the amino acid sequence of the antibody suchthat the number and position of cysteine residues is modified forcontrol of the number or position of linker-effector moleculeattachments.

C. Chimeric Antigen Receptors (CARs)

Also disclosed herein are chimeric antigen receptor (CARs) that areartificially constructed chimeric proteins including an extracellularantigen binding domain (e.g., single chain variable fragment (scFv))that specifically binds to IL-7Rα, linked to a transmembrane domain,linked to one or more intracellular T cell signaling domains.Characteristics of the disclosed CARs include their ability to redirectT cell specificity and reactivity towards IL-7Rα expressing cells in anon-MHC-restricted manner. The non-MHC-restricted IL-7Rα recognitiongives T cells expressing a disclosed CAR the ability to recognizeantigen independent of antigen processing.

The intracellular T cell signaling domains can include, for example, a Tcell receptor signaling domain, a T cell costimulatory signaling domain,or both. The T cell receptor signaling domain refers to a portion of theCAR comprising the intracellular domain of a T cell receptor, such asthe intracellular portion of the CD3 zeta protein. The costimulatorysignaling domain refers to a portion of the CAR comprising theintracellular domain of a costimulatory molecule, which is a cellsurface molecule other than an antigen receptor or their ligands thatare required for an efficient response of lymphocytes to antigen.

1. Extracellular Region

Several embodiments provide a CAR including an antigen binding domainthat specifically binds to IL-7Rα as disclosed herein (see, e.g.,section III.A). For example, the antigen binding domain can be a scFvincluding the V_(H) and the V_(L) of any of the 4A10 or 2B8 antibody, ora humanized or chimeric version thereof. In some embodiment, the antigenbinding domain can include a V_(H) and a V_(L) including the HCDR1,HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of the V_(H) and V_(L),respectively, of the 4A10 or the 2B8 antibody (e.g., as set forth inTable 1).

In some embodiments, the antigen binding domain includes a V_(H) and aV_(L) including the amino acid sequences set forth as SEQ ID NOs: 1 and2, respectively; or SEQ ID NOs: 3 and 4, respectively. In severalembodiments, the antigen binding domain can be a scFv. In someembodiments, the scFv includes a V_(H) and a V_(L) joined by a peptidelinker, such as a linker including the amino acid sequence set forth asGGGGSGGGGSGGGGS (SEQ ID NO: 37).

The CAR can include a signal peptide sequence, e.g., N-terminal to theantigen binding domain. The signal peptide sequence may comprise anysuitable signal peptide sequence. In an embodiment, the signal peptidesequence is a human granulocyte-macrophage colony-stimulating factor(GM-CSF) receptor sequence, such as an amino acid sequence including orconsisting of LLVTSLLLCELPHPAFLLIPDT (SEQ ID NO: 38). While the signalpeptide sequence may facilitate expression of the CAR on the surface ofthe cell, the presence of the signal peptide sequence in an expressedCAR is not necessary in order for the CAR to function. Upon expressionof the CAR on the cell surface, the signal peptide sequence may becleaved off of the CAR. Accordingly, in some embodiments, the CAR lacksa signal peptide sequence.

Between the antigen binding domain and the transmembrane domain of theCAR, there may be a spacer domain, which includes a polypeptidesequence. The spacer domain may comprise up to 300 amino acids,preferably 10 to 100 amino acids and most preferably 25 to 50 aminoacids. In some embodiments, the spacer domain can include animmunoglobulin domain, such as a human immunoglobulin sequence. In anembodiment, the immunoglobulin domain comprises an immunoglobulin CH2and CH3 immunoglobulin G (IgG1) domain sequence (CH2CH3). In thisregard, the spacer domain can include an immunoglobulin domaincomprising or consisting of the amino acid sequence set forth as SEQ IDNO: 39:

EPKSCDKTHTCPPCPAPELLGGPSVFLEPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKKDPKWithout being bound to a particular theory, it is believed that theCH2CH3 domain extends the antigen binding domain of the CAR away fromthe membrane of CAR-expressing cells and may more accurately mimic thesize and domain structure of a native TCR.

2. Transmembrane Domain

With respect to the transmembrane domain, the CAR can be designed tocomprise a transmembrane domain that is fused to the extracellulardomain of the CAR. In one embodiment, the transmembrane domain thatnaturally is associated with one of the domains in the CAR is used.

The transmembrane domain may be derived either from a natural or from asynthetic source. Where the source is natural, the domain may be derivedfrom any membrane-bound or transmembrane protein. Exemplarytransmembrane domains for use in the disclosed CARs can include at leastthe transmembrane region(s) of) the alpha, beta or zeta chain of the Tcell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CDS, CD9, CD 16, CD22,CD33, CD37, CD64, CD80, CD86, CD 134, CD137, CD154. Alternatively thetransmembrane domain may be synthetic, in which case it will comprisepredominantly hydrophobic residues such as leucine and valine. Inseveral embodiments, a triplet of phenylalanine, tryptophan and valinewill be found at each end of a synthetic transmembrane domain.

Optionally, a short oligo- or polypeptide linker, preferably between 2and 10 amino acids in length may form the linkage between thetransmembrane domain and the intracellular T cell signaling domainand/or T cell costimulatory domain of the CAR. An exemplary linkersequence includes one or more glycine-serine doublets.

In some embodiments, the transmembrane domain comprises thetransmembrane domain of a T cell receptor, such as a CD8 transmembranedomain. Thus, the CAR can include a CD8 transmembrane domain includingor consisting of SEQ ID NO: 40:

TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYI WAPLAGTCGVLLLSLVITLYCIn another embodiment, the transmembrane domain comprises thetransmembrane domain of a T cell costimulatory molecule, such as CD137or CD28. Thus, the CAR can include a CD28 transmembrane domain includingor consisting of SEQ ID NO: 41:

IEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKPFWVLVVVGG VLACYSLLVTVAFIIFWVR

3. Intracellular Region

The intracellular region of the CAR includes one or more intracellular Tcell signaling domains responsible for activation of at least one of thenormal effector functions of a T cell in which the CAR is expressed orplaced in. Exemplary T cell signaling domains are provided herein, andare known to the person of ordinary skill in the art.

While an entire intracellular T cell signaling domain can be employed ina CAR, in many cases it is not necessary to use the entire chain. To theextent that a truncated portion of the intracellular T cell signalingdomain is used, such truncated portion may be used in place of theintact chain as long as it transduces the relevant T cell effectorfunction signal.

Examples of intracellular T cell signaling domains for use in the CARinclude the cytoplasmic sequences of the T cell receptor (TCR) andco-stimulatory molecules that act in concert to initiate signaltransduction following antigen receptor engagement, as well as anyderivative or variant of these sequences and any synthetic sequence thathas the same functional capability.

T cell receptor signaling domains regulate primary activation of the Tcell receptor complex either in a stimulatory way, or in an inhibitoryway. The disclosed CARs can include primary cytoplasmic signalingsequences that act in a stimulatory manner, which may contain signalingmotifs that are known as immunoreceptor tyrosine-based activation motifsor ITAMs. Examples of ITAM containing primary cytoplasmic signalingsequences that can be included in a disclosed CAR include those from CD3zeta, FcR gamma, FcR beta, CD3 gamma , CD3 delta , CD3 epsilon, CDS,CD22, CD79a, CD79b, and CD66d proteins. In several embodiments, thecytoplasmic signaling molecule in the CAR includes an intracellular Tcell signaling domain from CD3 zeta.

The intracellular region of the CAR can include the ITAM containingprimary cytoplasmic signaling domain (such as CD3-zeta) by itself orcombined with any other desired cytoplasmic domain(s) useful in thecontext of a CAR. For example, the cytoplasmic domain of the CAR caninclude a CD3 zeta chain portion and an intracellular costimulatorysignaling domain. The costimulatory signaling domain refers to a portionof the CAR comprising the intracellular domain of a costimulatorymolecule. A costimulatory molecule is a cell surface molecule other thanan antigen receptor or their ligands that is required for an efficientresponse of lymphocytes to an antigen. Examples of such moleculesinclude CD27, CD28, 4-1BB (CD137), OX40 (CD134), CD30, CD40, PD-1, ICOS,lymphocyte function-associated antigen 1 (LFA-1), CD2, CD7, LIGHT,NKG2C, and B7-H3. An additional example of a signaling domain that canbe included in a disclosed CARs is a Tumor necrosis factor receptorsuperfamily member 18 (TNFRSF18; also known as glucocorticoid-inducedTNFR-related protein, GITR) signaling domain.

In some embodiments, the CAR can include a CD3 zeta signaling domain, aCD8 signaling domain, a CD28 signaling domain, a CD137 signaling domainor a combination of two or more thereof. In one embodiment, thecytoplasmic domain includes the signaling domain of CD3-zeta and thesignaling domain of CD28. In another embodiment, the cytoplasmic domainincludes the signaling domain of CD3 zeta and the signaling domain ofCD137. In yet another embodiment, the cytoplasmic domain includes thesignaling domain of CD3-zeta and the signaling domain of CD28 and CD137.The order of the one or more T cell signaling domains on the CAR can bevaried as needed by the person of ordinary skill in the art.

Exemplary amino acid sequences for such T cell signaling domains areprovided. For example, the CD3 zeta signaling domain can include orconsist of the amino acid sequence set forth as SEQ ID NO: 42(RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR), the CD8signaling domain can include or consist of the amino acid sequence setforth as SEQ ID NO: 43(FVPVFLPAKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCNHRNR), the CD28 signaling domain can include orconsist of the amino acid sequence set forth as SEQ ID NO: 44(SKRSRLLHSDYMNMTPRRPGPTRKHYQPYA PPRDFAAYRS), the CD137 signaling domaincan include or consist of the amino acid sequences set forth as SEQ IDNO: 45 (KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGC EL) or SEQ ID NO: 46(RFSVVKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL).

The cytoplasmic signaling sequences within the cytoplasmic signalingportion of the CAR of the invention may be linked to each other in arandom or specified order. Optionally, a short polypeptide linker,preferably between 2 and 10 amino acids in length may form the linkage.A glycine-serine doublet provides a particularly suitable linker.Further, between the signaling domain and the transmembrane domain ofthe CAR, there may be a spacer domain, which includes a polypeptidesequence. The spacer domain may comprise up to 300 amino acids,preferably 10 to 100 amino acids and most preferably 25 to 50 aminoacids.

4. Additional Description of CARs

Also provided are functional portions of the CARs described herein. Theterm “functional portion” when used in reference to a CAR refers to anypart or fragment of the CAR, which part or fragment retains thebiological activity of the CAR of which it is a part (the parent CAR).Functional portions encompass, for example, those parts of a CAR thatretain the ability to recognize target cells, or detect, treat, orprevent a disease, to a similar extent, the same extent, or to a higherextent, as the parent CAR. In reference to the parent CAR, thefunctional portion can comprise, for instance, about 10%, 25%, 30%, 50%,68%, 80%, 90%, 95%, or more, of the parent CAR.

The CAR or functional portion thereof, can include additional aminoacids at the amino or carboxy terminus, or at both termini, whichadditional amino acids are not found in the amino acid sequence of theparent CAR. Desirably, the additional amino acids do not interfere withthe biological function of the CAR or functional portion, e.g.,recognize target cells, detect cancer, treat or prevent cancer, etc.More desirably, the additional amino acids enhance the biologicalactivity, as compared to the biological activity of the parent CAR.

Also provided are functional variants of the CARs described herein,which have substantial or significant sequence identity or similarity toa parent CAR, which functional variant retains the biological activityof the CAR of which it is a variant. Functional variants encompass, forexample, those variants of the CAR described herein (the parent CAR)that retain the ability to recognize target cells to a similar extent,the same extent, or to a higher extent, as the parent CAR. In referenceto the parent CAR, the functional variant can, for instance, be at leastabout 30%, about 50%, about 75%, about 80%, about 85%, about 90%, about91%, about 92%, about 93%, about 94%, about 95%, about 96%), about 97%,about 98%, about 99% or more identical in amino acid sequence to theparent CAR.

A functional variant can, for example, comprise the amino acid sequenceof the parent CAR with at least one conservative amino acidsubstitution. Alternatively or additionally, the functional variants cancomprise the amino acid sequence of the parent CAR with at least onenon-conservative amino acid substitution. In this case, it is preferablefor the non-conservative amino acid substitution to not interfere withor inhibit the biological activity of the functional variant. Thenon-conservative amino acid substitution may enhance the biologicalactivity of the functional variant, such that the biological activity ofthe functional variant is increased as compared to the parent CAR.

The CARs (including functional portions and functional variants) can beof any length, i.e., can comprise any number of amino acids, providedthat the CARs (or functional portions or functional variants thereof)retain their biological activity, e.g., the ability to specifically bindto antigen, detect diseased cells in a mammal, or treat or preventdisease in a mammal, etc. For example, the CAR can be about 50 to about5000 amino acids long, such as 50, 70, 75, 100, 125, 150, 175, 200, 300,400, 500, 600, 700, 800, 900, 1000 or more amino acids in length.

The CARs (including functional portions and functional variants of theinvention) can comprise synthetic amino acids in place of one or morenaturally-occurring amino acids. Such synthetic amino acids are known inthe art, and include, for example, aminocyclohexane carboxylic acid,norleucine, a-amino n-decanoic acid, homoserine,S-acetylaminomethyl-cysteine, trans-3- and trans-4-hydroxyproline,4-aminophenylalanine, 4-nitrophenylalanine, 4-chlorophenylalanine,4-carboxyphenylalanine, β-phenylserine β-hydroxyphenylalanine,phenylglycine, α-naphthylalanine, cyclohexylalanine, cyclohexylglycine,indoline-2-carboxylic acid, 1,2,3,4-tetrahydroisoquinoline-3-carboxylicacid, aminomalonic acid, aminomalonic acid monoamide,N′-benzyl-N′-methyl-lysine, N′,N′-dibenzyl-lysine, 6-hydroxylysine,ornithine, a-aminocyclopentane carboxylic acid, a-aminocyclohexanecarboxylic acid, oc-aminocycloheptane carboxylic acid,-(2-amino-2-norbornane)-carboxylic acid, γ-diaminobutyric acid,α,β-diaminopropionic acid, homophenylalanine, and α-tert-butylglycine.

The CARs (including functional portions and functional variants) can beglycosylated, amidated, carboxylated, phosphorylated, esterified,N-acylated, cyclized via, e.g., a disulfide bridge, or converted into anacid addition salt and/or optionally dimerized or polymerized, orconjugated.

Methods of generating chimeric antigen receptors, T cells including suchreceptors, and their use (e.g., for treatment of cancer) are known inthe art and further described herein (see, e.g., Brentjens et al., 2010,Molecular Therapy, 18:4, 666-668; Morgan et al., 2010, MolecularTherapy, published online Feb. 23, 2010, pages 1 -9; Till et al., 2008,Blood, 1 12:2261 -2271; Park et al., Trends Biotechnol., 29:550-557,2011; Grupp et al., N Engl J Med., 368:1509-1518, 2013; Han et al., J.Hematol Oncol., 6:47, 2013; PCT Pub. WO02012/079000, WO2013/126726; andU.S. Pub. 2012/0213783, each of which is incorporated by referenceherein in its entirety.) For example, a nucleic acid molecule encoding adisclosed chimeric antigen binding receptor can be included in anexpression vector (such as a lentiviral vector) for expression in a hostcell, such as a T cell, to make the disclosed CAR. In some embodiments,methods of using the chimeric antigen receptor include isolating T cellsfrom a subject, transforming the T cells with an expression vector (suchas a lentiviral vector) encoding the chimeric antigen receptor, andadministering the engineered T cells expressing the chimeric antigenreceptor to the subject for treatment, for example for treatment of aIL-7Rα-positive cancer in the subject.

D. Polynucleotides and Expression

Nucleic acids molecules (for example, cDNA molecules) encoding the aminoacid sequences of antibodies, antibody binding fragments, CARs andconjugates that specifically bind IL-7Rα are provided. Nucleic acidsencoding these molecules can readily be produced by one of skill in theart, using the amino acid sequences provided herein (such as the CDRsequences and V_(H) and V_(L) sequences), sequences available in the art(such as framework or constant region sequences), and the genetic code.In several embodiments, a nucleic acid molecules can encode the V_(H),the V_(L), or both the V_(H) and V_(L) (for example in a bicistronicexpression vector) of a disclosed antibody or antigen binding fragment,or a humanized version thereof. In several embodiments, the nucleic acidmolecules can be expressed in a host cell (such as a mammalian cell) toproduce a disclosed antibody or antigen binding fragment.

One of skill in the art can readily use the genetic code to construct avariety of functionally equivalent nucleic acids, such as nucleic acidswhich differ in sequence but which encode the same antibody sequence, orencode a conjugate or fusion protein including the V_(L) and/or V_(H)nucleic acid sequence.

In a non-limiting example, an isolated nucleic acid molecule encodes theV_(H) of a disclosed antibody or antigen binding fragment and includesthe nucleic acid sequence set forth as any one of SEQ ID NOs: 25 or 27.In a non-limiting example, an isolated nucleic acid molecule encodes theV_(L) of a disclosed antibody or antigen binding fragment and includesthe nucleic acid sequence set forth as any one of SEQ ID NOs: 26 or 28.In a non-limiting example, an isolated nucleic acid molecule encodes theV_(H) and V_(L) of a disclosed antibody or antigen binding fragment andincludes the nucleic acid sequences set forth as any one of SEQ ID NOs:25 and 26, respectively, or 27 and 28, respectively.

Nucleic acid sequences encoding the antibodies, antibody bindingfragments, CARs and conjugates that specifically bind IL-7Rα can beprepared by any suitable method including, for example, cloning ofappropriate sequences or by direct chemical synthesis by methods such asthe phosphotriester method of Narang et al., Meth. Enzymol. 68:90-99,1979; the phosphodiester method of Brown et al., Meth. Enzymol.68:109-151, 1979; the diethylphosphoramidite method of Beaucage et al.,Tetra. Lett. 22:1859-1862, 1981; the solid phase phosphoramiditetriester method described by Beaucage & Caruthers, Tetra. Letts.22(20):1859-1862, 1981, for example, using an automated synthesizer asdescribed in, for example, Needham-VanDevanter et al., Nucl. Acids Res.12:6159-6168, 1984; and, the solid support method of U.S. Pat. No.4,458,066. Chemical synthesis produces a single strandedoligonucleotide. This can be converted into double stranded DNA byhybridization with a complementary sequence or by polymerization with aDNA polymerase using the single strand as a template.

Exemplary nucleic acids can be prepared by cloning techniques. Examplesof appropriate cloning and sequencing techniques, and instructionssufficient to direct persons of skill through many cloning exercises areknown (see, e.g, Sambrook et al. (Molecular Cloning: A LaboratoryManual, 4^(th) ed, Cold Spring Harbor, N.Y., 2012) and Ausubel et al.(In Current Protocols in Molecular Biology, John Wiley & Sons, New York,through supplement 104, 2013). Product information from manufacturers ofbiological reagents and experimental equipment also provide usefulinformation. Such manufacturers include the SIGMA Chemical Company(Saint Louis, Mo.), R&D Systems (Minneapolis, Minn.), Pharmacia Amersham(Piscataway, N.J.), CLONTECH Laboratories, Inc. (Palo Alto, Calif.),Chem Genes Corp., Aldrich Chemical Company (Milwaukee, Wis.), GlenResearch, Inc., GIBCO BRL Life Technologies, Inc. (Gaithersburg, Md.),Fluka Chemica-Biochemika Analytika (Fluka Chemie AG, Buchs,Switzerland), Invitrogen (Carlsbad, Calif.), and Applied Biosystems(Foster City, Calif.), as well as many other commercial sources known toone of skill.

Nucleic acids can also be prepared by amplification methods.Amplification methods include polymerase chain reaction (PCR), theligase chain reaction (LCR), the transcription-based amplificationsystem (TAS), the self-sustained sequence replication system (3SR). Awide variety of cloning methods, host cells, and in vitro amplificationmethodologies are well known to persons of skill.

In some embodiments, the nucleic acid molecule encodes a CAR as providedherein for expression in a T cell to generate a chimeric antigenreceptor T cell. The nucleic acid molecule encoding the chimeric antigenbinding receptor can be included in a vector (such as a lentiviralvector) for expression in a host cell, such as a T cell. Exemplary cellsinclude a T cell, a Natural Killer (NK) cell, a cytotoxic T lymphocyte(CTL), and a regulatory T cell. Methods of generating nucleic acidmolecules encoding chimeric antigen receptors and T cells including suchreceptors are known in the art (see, e.g., Brentjens et al., 2010,Molecular Therapy, 18:4, 666-668; Morgan et al., 2010, MolecularTherapy, published online Feb. 23, 2010, pages 1 -9; Till et al., 2008,Blood, 1 12:2261 -2271; Park et al., Trends Biotechnol., 29:550-557,2011; Grupp et al., N Engl J Med., 368:1509-1518, 2013; Han et al., J.Hematol Oncol., 6:47, 2013; PCT Pub. WO2012/079000, WO2013/126726; andU.S. Pub. 2012/0213783, each of which is incorporated by referenceherein in its entirety.)

The nucleic acid molecules can be expressed in a recombinantlyengineered cell such as bacteria, plant, yeast, insect and mammaliancells. The antibodies, antigen binding fragments, and conjugates can beexpressed as individual V_(H) and/or V_(L) chain (linked to an effectormolecule or detectable marker as needed), or can be expressed as afusion protein. Methods of expressing and purifying antibodies andantigen binding fragments are known and further described herein (see,e.g., Al-Rubeai (ed), Antibody Expression and Production, SpringerPress, 2011). An immunoadhesin can also be expressed. Thus, in someexamples, nucleic acids encoding a V_(H) and V_(L), and immunoadhesinare provided. The nucleic acid sequences can optionally encode a leadersequence.

To create a scFv the V_(H)- and V_(L)-encoding DNA fragments can beoperatively linked to another fragment encoding a flexible linker, e.g.,encoding the amino acid sequence (Gly₄-Ser)₃, such that the V_(H) andV_(L) sequences can be expressed as a contiguous single-chain protein,with the V_(L) and V_(H) domains joined by the flexible linker (see,e.g., Bird et al., Science 242:423-426, 1988; Huston et al., Proc. Natl.Acad. Sci. USA 85:5879-5883, 1988; McCafferty et al., Nature348:552-554, 1990; Kontermann and Dubel (Ed), Antibody Engineering,Vols. 1-2, 2^(nd) Ed., Springer Press, 2010; Harlow and Lane,Antibodies: A Laboratory Manual, 2^(nd), Cold Spring Harbor Laboratory,New York, 2013,). Optionally, a cleavage site can be included in alinker, such as a furin cleavage site.

The single chain antibody may be monovalent, if only a single V_(H) andV_(L) are used, bivalent, if two V_(H) and V_(L) are used, orpolyvalent, if more than two V_(H) and V_(L) are used. Bispecific orpolyvalent antibodies may be generated that bind specifically to IL-7Rαand another antigen, such as, but not limited to CD3. The encoded V_(H)and V_(L) optionally can include a furin cleavage site between the V_(H)and V_(L) domains.

The nucleic acid encoding a V_(H) and/or the V_(L) optionally can encodean Fc domain (immunoadhesin). The Fc domain can be an IgA, IgM or IgG Fcdomain. The Fc domain can be an optimized Fc domain, as described inU.S. Published Patent Application No. 20100/093979, incorporated hereinby reference. In one example, the immunoadhesin is an IgG₁ Fc.

Those of skill in the art are knowledgeable in the numerous expressionsystems available for expression of proteins including E. coli, otherbacterial hosts, yeast, and various higher eukaryotic cells such as theCOS, CHO, HeLa and myeloma cell lines.

One or more DNA sequences encoding the antibodies, antibody bindingfragments, CARs or conjugates can be expressed in vitro by DNA transferinto a suitable host cell. The cell may be prokaryotic or eukaryotic.The term also includes any progeny of the subject host cell. It isunderstood that all progeny may not be identical to the parental cellsince there may be mutations that occur during replication. Methods ofstable transfer, meaning that the foreign DNA is continuously maintainedin the host, are known in the art. Hybridomas expressing the antibodiesof interest are also encompassed by this disclosure.

The expression of nucleic acids encoding the antibodies and antigenbinding fragments described herein can be achieved by operably linkingthe DNA or cDNA to a promoter (which is either constitutive orinducible), followed by incorporation into an expression cassette. Thepromoter can be any promoter of interest, including a cytomegaloviruspromoter and a human T cell lymphotrophic virus promoter (HTLV)-1.Optionally, an enhancer, such as a cytomegalovirus enhancer, is includedin the construct. The cassettes can be suitable for replication andintegration in either prokaryotes or eukaryotes. Typical expressioncassettes contain specific sequences useful for regulation of theexpression of the DNA encoding the protein. For example, the expressioncassettes can include appropriate promoters, enhancers, transcriptionand translation terminators, initiation sequences, a start codon (i.e.,ATG) in front of a protein-encoding gene, splicing signal for introns,sequences for the maintenance of the correct reading frame of that geneto permit proper translation of mRNA, and stop codons. The vector canencode a selectable marker, such as a marker encoding drug resistance(for example, ampicillin or tetracycline resistance).

To obtain high level expression of a cloned gene, it is desirable toconstruct expression cassettes which contain, at the minimum, a strongpromoter to direct transcription, a ribosome binding site fortranslational initiation (internal ribosomal binding sequences), and atranscription/translation terminator. For E. coli, this can include apromoter such as the T7, trp, lac, or lambda promoters, a ribosomebinding site, and preferably a transcription termination signal. Foreukaryotic cells, the control sequences can include a promoter and/or anenhancer derived from, for example, an immunoglobulin gene, HTLV, SV40or cytomegalovirus, and a polyadenylation sequence, and can furtherinclude splice donor and/or acceptor sequences (for example, CMV and/orHTLV splice acceptor and donor sequences). The cassettes can betransferred into the chosen host cell by well-known methods such astransformation or electroporation for E. coli and calcium phosphatetreatment, electroporation or lipofection for mammalian cells. Cellstransformed by the cassettes can be selected by resistance toantibiotics conferred by genes contained in the cassettes, such as theamp, gpt, neo and hyg genes.

When the host is a eukaryote, such methods of transfection of DNA ascalcium phosphate coprecipitates, conventional mechanical proceduressuch as microinjection, electroporation, insertion of a plasmid encasedin liposomes, or virus vectors may be used Eukaryotic cells can also becotransformed with polynucleotide sequences encoding the antibody,labeled antibody, or antigen biding fragment, and a second foreign DNAmolecule encoding a selectable phenotype, such as the herpes simplexthymidine kinase gene. Another method is to use a eukaryotic viralvector, such as simian virus 40 (SV40) or bovine papilloma virus, totransiently infect or transform eukaryotic cells and express the protein(see for example, Viral Expression Vectors, Springer press, Muzyczkaed., 2011). One of skill in the art can readily use an expressionsystems such as plasmids and vectors of use in producing proteins incells including higher eukaryotic cells such as the COS, CHO, HeLa andmyeloma cell lines.

For purposes of producing a recombinant CAR, the host cell may be amammalian cell. The host cell may be a human cell. In some embodiments,the host cell may be a peripheral blood lymphocyte (PBL) or a peripheralblood mononuclear cell (PBMC), or a T cell. The T cell can be any Tcell, such as a cultured T cell, e.g., a primary T cell, or a T cellfrom a cultured T cell line, e.g., Jurkat, SupT1, etc., or a T cellobtained from a mammal (such as a human patient to which the CAR-T cellwill later be administered). If obtained from a mammal, the T cell canbe obtained from numerous sources, including but not limited to blood,bone marrow, lymph node, the thymus, or other tissues or fluids. T cellscan also be enriched for or purified. The T cell may be a human T cell.The T cell may be a T cell isolated from a human. The T cell can be anytype of T cell and can be of any developmental stage, including but notlimited to, CD4⁺/CD8⁺ double positive T cells, CD4⁺ helper T cells,e.g., Th₁ and Th₂ cells, CD8⁺ T cells (e.g., cytotoxic T cells), tumorinfiltrating cells, memory T cells, naive T cells, and the like. The Tcell may be a CD8⁺ T cell or a CD4⁺ T cell.

Also provided is a population of cells comprising at least one host celldescribed herein. The population of cells can be a heterogeneouspopulation comprising the host cell comprising any of the recombinantexpression vectors described, in addition to at least one other cell,e.g., a host cell (e.g., a T cell), which does not comprise any of therecombinant expression vectors, or a cell other than a T cell, e.g., a Bcell, a macrophage, a neutrophil, an erythrocyte, a hepatocyte, anendothelial cell, an epithelial cell, a muscle cell, a brain cell, etc.Alternatively, the population of cells can be a substantiallyhomogeneous population, in which the population comprises mainly hostcells (e.g., consisting essentially of) comprising the recombinantexpression vector. The population also can be a clonal population ofcells, in which all cells of the population are clones of a single hostcell comprising a recombinant expression vector, such that all cells ofthe population comprise the recombinant expression vector. In oneembodiment of the invention, the population of cells is a clonalpopulation comprising host cells comprising a recombinant expressionvector as described herein

Modifications can be made to a nucleic acid encoding a polypeptidedescribed herein without diminishing its biological activity. Somemodifications can be made to facilitate the cloning, expression, orincorporation of the targeting molecule into a fusion protein. Suchmodifications are well known to those of skill in the art and include,for example, termination codons, a methionine added at the aminoterminus to provide an initiation, site, additional amino acids placedon either terminus to create conveniently located restriction sites, oradditional amino acids (such as poly His) to aid in purification steps.In addition to recombinant methods, the immunoconjugates, effectormoieties, and antibodies of the present disclosure can also beconstructed in whole or in part using standard peptide synthesis wellknown in the art.

Once expressed, the antibodies, antigen binding fragments, andconjugates can be purified according to standard procedures in the art,including ammonium sulfate precipitation, affinity columns, columnchromatography, and the like (see, generally, Simpson ed., Basic methodsin Protein Purification and Analysis: A laboratory Manual, Cold HarborPress, 2008). The antibodies, antigen binding fragment, and conjugatesneed not be 100% pure. Once purified, partially or to homogeneity asdesired, if to be used therapeutically, the polypeptides should besubstantially free of endotoxin.

Methods for expression of the antibodies, antigen binding fragments, andconjugates, and/or refolding to an appropriate active form, frommammalian cells, and bacteria such as E. coli have been described andare well-known and are applicable to the antibodies disclosed herein.See, e.g., Harlow and Lane, Antibodies: A Laboratory Manual, 2^(nd),Cold Spring Harbor Laboratory, New York, 2013, Simpson ed., Basicmethods in Protein Purification and Analysis: A laboratory Manual, ColdHarbor Press, 2008, and Ward et al., Nature 341:544, 1989.

E. Methods and Compositions 1. Therapeutic Methods Cancer

The IL-7Rα-specific antibodies, antigen binding fragments, conjugates,and CAR T cells disclosed herein can be administered a subject to slowor inhibit the growth of cancer cells expressing IL-7Rα, such asproliferating T or B cells. In these applications, an IL-7Rα-specificantibody, antigen binding fragment, conjugate, or CAR T cell asdisclosed herein is administered to a subject in an amount sufficient toinhibit growth, replication, or metastasis of the cancer cellsexpressing IL-7Rα, or to inhibit a sign or a symptom of the cancer.Suitable subjects include those diagnosed with an IL-7Rα-positive cancer(that is, a cancer including cells that expresses IL-7Rα protein), suchas, but not limited to, ALL, for example, T-ALL or B-ALL.

In one non-limiting embodiment, provided herein is a method of treatinga subject with an IL-7R-positive cancer by selecting a subject having acancer that expresses IL-7Rα protein and administering to the subject atherapeutically effective amount of an IL-7Rα-specific antibody, antigenbinding fragment, conjugate, or CAR T cell as disclosed herein. Alsoprovided herein is a method of inhibiting growth or metastasis ofIL-7Rα-positive cancer cells by selecting a subject having anIL-7Rα-positive cancer and administering to the subject atherapeutically effective amount of an IL-7Rα-specific antibody, antigenbinding fragment, conjugate, or CAR T cell as disclosed herein.

A therapeutically effective amount of an IL-7Rα-specific antibody,antigen binding fragment, conjugate, or CAR T cell as disclosed hereinwill depend upon the severity of the disease and the general state ofthe patient's health. A therapeutically effective amount is that whichprovides either subjective relief of a symptom(s) or an objectivelyidentifiable improvement as noted by the clinician or other qualifiedobserver. In some examples, therapeutic amounts are amounts whicheliminate or reduce the patient's tumor burden, or which prevent orreduce the proliferation of metastatic cells (such as IL-7Rα-positivecancer cells).

Subjects that can benefit from the disclosed methods include human andveterinary subjects. Subjects can be screened prior to initiating thedisclosed therapies, for example to determine whether the subject has anIL-7Rα-positive cancer such as ALL. The presence of the IL-7Rα cancerindicates that the subject can be treated using the methods providedherein.

Administration of the antibodies, antigen binding fragments, conjugates,CAR T cells, or compositions can be accompanied by administration ofother anti-cancer agents or therapeutic treatments (such as surgicalresection of a tumor or radiation therapy). Any suitable anti-canceragent can be administered in combination with the antibodies andconjugates disclosed herein. Exemplary anti-cancer agents include, butare not limited to, chemotherapeutic agents, such as, for example,mitotic inhibitors, alkylating agents, anti-metabolites, intercalatingantibiotics, growth factor inhibitors, cell cycle inhibitors, enzymes,topoisomerase inhibitors, anti-survival agents, biological responsemodifiers, anti-hormones (e.g. anti-androgens) and anti-angiogenesisagents. Other anti-cancer treatments include radiation therapy and otherantibodies that specifically target cancer cells.

Non-limiting examples of alkylating agents include nitrogen mustards(such as mechlorethamine, cyclophosphamide, melphalan, uracil mustard orchlorambucil), alkyl sulfonates (such as busulfan), nitrosoureas (suchas carmustine, lomustine, semustine, streptozocin, or dacarbazine).

Non-limiting examples of antimetabolites include folic acid analogs(such as methotrexate), pyrimidine analogs (such as 5-FU or cytarabine),and purine analogs, such as mercaptopurine or thioguanine.

Non-limiting examples of natural products include vinca alkaloids (suchas vinblastine, vincristine, or vindesine), epipodophyllotoxins (such asetoposide or teniposide), antibiotics (such as dactinomycin,daunorubicin, doxorubicin, bleomycin, plicamycin, or mitomycin C), andenzymes (such as L-asparaginase).

Non-limiting examples of miscellaneous agents include platinumcoordination complexes (such as cis-diamine-dichloroplatinum II alsoknown as cisplatin), substituted ureas (such as hydroxyurea), methylhydrazine derivatives (such as procarbazine), and adrenocroticalsuppressants (such as mitotane and aminoglutethimide).

Non-limiting examples of hormones and antagonists includeadrenocorticosteroids (such as prednisone, prednisolone), progestins(such as hydroxyprogesterone caproate, medroxyprogesterone acetate, andmagestrol acetate), estrogens (such as diethylstilbestrol and ethinylestradiol), antiestrogens (such as tamoxifen), and androgens (such astesterone proprionate and fluoxymesterone).

Examples of the most commonly used chemotherapy drugs includeAdriamycin, Alkeran, Ara-C, BiCNU, Busulfan, CCNU, Carboplatinum,Cisplatinum, Cytoxan, Daunorubicin, DTIC, 5-FU, Fludarabine, Hydrea,Idarubicin, Ifosfamide, Methotrexate, Mithramycin, Mitomycin,Mitoxantrone, Nitrogen Mustard, Taxol (or other taxanes, such asdocetaxel), Velban, Vincristine, VP-16, while some more newer drugsinclude Gemcitabine (Gemzar), Herceptin®, Irinotecan (Camptosar,CPT-11), Leustatin, Navelbine, Rituxan STI-571, Taxotere, Topotecan(Hycamtin), Xeloda (Capecitabine), Zevelin and calcitriol.

Non-limiting examples of immunomodulators that can be used includeAS-101 (Wyeth-Ayerst Labs.), bropirimine (Upjohn), gamma interferon(Genentech), GM-CSF (granulocyte macrophage colony stimulating factor;Genetics Institute), IL-2 (Cetus or Hoffman-LaRoche), human immuneglobulin (Cutter Biological), IMREG (from Imreg of New Orleans, La.),SK&F 106528, and TNF (tumor necrosis factor; Genentech).

Another common treatment for some types of cancer is surgical treatment,for example surgical resection of the cancer or a portion of it. Anotherexample of a treatment is radiotherapy, for example administration ofradioactive material or energy (such as external beam therapy) to thecancer site to help eradicate the cancer or shrink it prior to surgicalresection.

In another aspect, a therapeutically effective amount of a combinationtherapy including administration of any of the IL-7Rα-specificantibodies, antigen binding fragments, conjugates, CAR T cells, orcompositions described herein with administration a CXCR4 antagonist canbe provided to a paitent. As described in the Examples, combinationtherapy including an IL-7Rα antibody and a CXCR4 antagonistsynergistically depleted T-ALL xenograft cells from bone marrow in amouse model of T-ALL. In some embodiments, the combination therapy caninclude the administration of an IL-7Rα-specific antibody, antigenbinding fragment, conjugate, or CAR T cell as described herein as wellas administration of the CXCR4 antagonist AMD3100 (plerixafor, GenzymeCorp.).

Accordingly, the combination therapy may provide synergy and provesynergistic, that is, the effect achieved when the active ingredientsused together is greater than the sum of the effects that results fromusing the compounds separately. A synergistic effect may be attainedwhen the active ingredients are: (1) co-formulated and administered ordelivered simultaneously in a combined, unit dosage formulation; (2)delivered by alternation or in parallel as separate formulations; or (3)by some other regimen. When delivered in alternation, a synergisticeffect may be attained when the compounds are administered or deliveredsequentially, for example by different injections in separate syringes.In general, during alternation, an effective dosage of each activeingredient is administered sequentially, i.e. serially, whereas incombination therapy, effective dosages of two or more active ingredientsare administered together. The synergy allows for reduced dosages of theactive agents in combination as compared to the dosages for eitheractive individually. The reduced dosage can help reduce any side effectsthat may appear. One of skill in medicine can best determine theappropriate dose of the additional therapeutic agent by considering thestate of the patient, the recommended dose, the severity of disease, andany synergistic effect of the combination.

In some examples, a subject is administered the DNA encoding theantibody or antigen binding fragments thereof, to provide in vivoantibody production, for example using the cellular machinery of thesubject. Immunization by nucleic acid constructs is well known in theart and taught, for example, in U.S. Pat. No. 5,643,578, and U.S. Pat.No. 5,593,972 and U.S. Pat. No. 5,817,637. U.S. Pat. No. 5,880,103describes several methods of delivery of nucleic acids encoding to anorganism. One approach to administration of nucleic acids is directadministration with plasmid DNA, such as with a mammalian expressionplasmid. The nucleotide sequence encoding the disclosed antibody, orantigen binding fragments thereof, can be placed under the control of apromoter to increase expression. The methods include liposomal deliveryof the nucleic acids. Such methods can be applied to the production ofan antibody, or antigen binding fragments thereof, by one of ordinaryskill in the art. In some embodiments, a disclosed antibody or antigenbinding fragment is expressed in a subject using the pVRC8400 vector(described in Barouch et al., J. Virol, 79 ,8828-8834, 2005, which isincorporated by reference herein).

The nucleic acid molecules encoding the disclosed antibodies or antigenbinding fragments can be included in a viral vector, for example forexpression of the antibody or antigen binding fragment in a host cell,or a subject (such as a subject with or at risk of T-ALL). A number ofviral vectors have been constructed, that can be used to express thedisclosed antibodies or antigen binding fragments, such as a retroviralvector, an adenoviral vector, or an adeno-associated virus (AAV) vector.In several examples, the viral vector can be replication-competent. Forexample, the viral vector can have a mutation in the viral genome thatdoes not inhibit viral replication in host cells. The viral vector alsocan be conditionally replication-competent. In other examples, the viralvector is replication-deficient in host cells.

In several embodiments, a subject (such as a human subject with or atrisk of T-ALL) can be administered a therapeutically effective amount ofan adeno-associated virus (AAV) viral vector that includes one or morenucleic acid molecules encoding a disclosed antibody or antigen bindingfragment. The AAV viral vector is designed for expression of the nucleicacid molecules encoding a disclosed antibody or antigen bindingfragment, and administration of the therapeutically effective amount ofthe AAV viral vector to the subject leads to expression of atherapeutically effective amount of the antibody or antigen bindingfragment in the subject. Non-limiting examples of AAV viral vectors thatcan be used to express a disclosed antibody or antigen binding fragmentin a subject include those provided in Johnson et al (“Vector-mediatedgene transfer engenders long-lived neutralizing activity and protectionagainst SIV infection in monkeys,” Nat. Med., 15(8):901-906, 2009) andGardner et al. (“AAV-expressed eCD4-Ig provides durable protection frommultiple SHIV challenges,” Nature, 519(7541): 87-91, 2015), each ofwhich is incorporated by reference herein in its entirety.

In one embodiment, a nucleic acid encoding a disclosed antibody, orantigen binding fragments thereof, is introduced directly into cells.For example, the nucleic acid can be loaded onto gold microspheres bystandard methods and introduced into the skin by a device such asBio-Rad's HELIOS™ Gene Gun. The nucleic acids can be “naked,” consistingof plasmids under control of a strong promoter.

Typically, the DNA is injected into muscle, although it can also beinjected directly into other sites. Dosages for injection are usuallyaround 0.5 μg/kg to about 50 mg/kg, and typically are about 0.005 mg/kgto about 5 mg/kg (see, e.g., U.S. Pat. No. 5,589,466).

Autoimmune Disorders

In additional embodiments, a method is provided for preventing ortreating an autoimmune disorder in a subject by administering to thesubject a therapeutically effective amount of an IL-7Rα specificantibody or antigen binding fragment as provided herein. Polymorphismswithin components of the IL-7 pathway are known to alter risk of anautoimmune disorder. For example, an allele encoding a threonine atposition 244 of IL-7Rα is a high risk allele for autoimmunity, but anallele encoding an isoleucine at the same position is a low risk allelefor autoimmunity. The position 244 polymorphism lies in the same regionin exon 6 identified for gain-of-function oncogenic mutations. Onenon-limiting explanation for the increase in autoimmunity risk due toparticular polymorphisms in the IL-7Rα gene is that these mutations maylead to increased IL-7R signaling activity. Accordingly, targeting theIL-7R pathway may have therapeutic benefit in numerous diseases withautoimmune or immune excess components. A non-limiting list ofautoimmune diseases for which an association of IL-7Rα polymorphism andautoimmunity risk has been shown is provided in the following table.

IL-7Rα Disease polymorphism reference multiple sclerosis T244I (exon 6)Hafler et al., N. Engl. J. Med., 357: 851-62, 2007 Gregory et al., Nat.Genet., 39: 1083-91, 2007 Lundmark et al., Neuroimmunol., 192: 171-73,2007 Type I diabetes T244I (exon 6) Todd et al, Nat. Genet., 39: 857-64,2007 Santiago et al, Diabetologia, 51: 1653-58, 2008 rheumatoidarthritis T244I (exon 6) O'Doherty et al, Tissue Antigens, 74: 429-31,2009 Sarcoidosis T244I (exon 6) Heron et al, Genes Immun., 10: 647-53,2009 Atopic dermatitis T244I (exon 6) and Hoffjan et al, J. DermatolSci., 55: 138-40, 2009 T46I (exon 2) Inhalation allergy T244I (exon 6)and Shamim et al, Int. J. Immunogenet., 34: 149-51, 2007 I118V (exon 4)graft versus host T46I (exon 2) and Shamim et al, Transplantation, 91:731-36, 2011 disease I118V (exon 4) Primary Biliary (possible T244I)Mells et al, Nat. Genet., 43: 329-32, 2011 Cirrhosis Inflammatory (non244) Anderson et al, Nat. Genet., 43: 246-52, 2011 bowel disease

Accordingly, in some embodiments, the disclosed antibodies and antigenbinding fragments can be used to treat or prevent an autoimmune disordersuch as multiple sclerosis, type 1 diabetes, rheumatoid arthritis,sarcoidosis, atopic dermatitis, inhalation allergy, primary biliarycirrhosis, or inflammatory bowel disease, in a subject, the methodcomprising administering to the subject a therapeutically effectiveamount of a disclosed antibody or antigen binding fragment thatspecifically binds to IL-7Rα. In some embodiments, the disclosedantibodies and antigen binding fragments can be used to treat or preventgraft versus host disease in a subject, the method comprisingadministering to the subject a therapeutically effective amount of adisclosed antibody or antigen binding fragment that specifically bindsto IL-7Rα.

In some embodiments, administration of the therapeutically effectiveamount of the IL-7Rα specific antibody or antigen binding fragment to asubject with type I diabetes can reduce or prevent one or more symptomsof the type I diabetes including, for example, a reduction in bloodglucose level and/or improved glucose tolerance in the subject.

In some embodiments, administration of the therapeutically effectiveamount of the IL-7Rα specific antibody or antigen binding fragment to asubject with rheumatoid arthritis can reduce or prevent one or moresymptoms of the rheumatoid arthritis including, for example, jointstiffness, joint swelling, joint pain, and/or joint redness and warmth.

In some embodiments, administration of the therapeutically effectiveamount of the L-7Rα specific antibody or antigen binding fragment to asubject with lupus can reduce or prevent one or more symptoms of thelupus including, for example, fatigue, fever, weight loss, weight gain,joint pain, joint stiffness, joint swelling, malar rash, skin lesions,mouth sores, nose ulcers, hair loss, Raynaud's phenomenon, shortness ofbreath, chest pain, dry eyes, bruising, anxiety, depression and memoryloss.

In some embodiments, administration of the therapeutically effectiveamount of the IL-7Rα specific antibody or antigen binding fragment to asubject with multiple sclerosis can reduce or prevent one or moresymptoms of the multiple sclerosis including, for example, limbparalysis, tremors, difficulty walking, swallowing difficulties,blindness, blurring vision, and muscle weakness.

In some embodiments, administration of the therapeutically effectiveamount of the -7Rα specific antibody or antigen binding fragment to asubject with graft versus host disease can reduce or prevent one or moresymptoms of the graft versus host disease including, for example,abdominal pain, abdominal cramps, fever, jaundice, skin rash, vomiting,weight loss, dry eyes, dry mouth, hair loss, hepatitis, lung disorders,and digestive tract disorders.

In some embodiments, administration of the therapeutically effectiveamount of the IL-7Rα, specific antibody or antigen binding fragment to asubject with acute graft versus host disease can reduce or prevent oneor more symptoms of the acute graft versus host disease including, forexample, pneumonitis, intestinal inflammation, diarrhea, abdominal pain,abdominal cramps, fever, jaundice, nausea, vomiting, liver damage, skinrash, skin damage, damage to the mucosa, sloughing of the mucosalmembrane, damage to the gastrointestinal tract, weight loss,maculopapular rash, elevated bilirubin levels, morbidity and mortality.

In some embodiments, administration of the therapeutically effectiveamount of the IL-7Rα specific antibody or antigen binding fragment to asubject with chronic graft versus host disease can reduce or prevent oneor more symptoms of the chronic graft versus host disease including, forexample, dry eyes, dry mouth, hair loss, hepatitis, lung disorders,digestive tract disorders, skin rash, oral ulcer, oral atrophy,onchodystrophy, sicca syndrome, sclerosis, lichen-planus-like lesions,poikiloderma, esophageal webs, fasciitis and bronchiolitis obliterans,and damage to the liver, skin and mucosa, connective tissue, exocrineglands and/or the gastrointestinal tract.

2. Dosages

A therapeutically effective amount of a IL-7Rα-specific antibody,antigen binding fragment, conjugate, CAR, T cell expressing a CAR, ornucleic acid molecule encoding such molecules, will depend upon theseverity of the disease and the general state of the patient's health. Atherapeutically effective amount is that which provides eithersubjective relief of a symptom(s) or an objectively identifiableimprovement as noted by the clinician or other qualified observer. TheIL-7Rα-specific antibody, antigen binding fragment, conjugate, CAR, Tcell expressing a CAR, or nucleic acid molecule encoding such molecules,can be administered in conjunction with another therapeutic agent,either simultaneously or sequentially.

Single or multiple administrations of a composition including adisclosed IL-7Rα-specific antibody, antigen binding fragment, conjugate,CAR, T cell expressing a CAR, or nucleic acid molecule encoding suchmolecules, can be administered depending on the dosage and frequency asrequired and tolerated by the patient. Compositions including theIL-7Rα-specific antibody, antigen binding fragment, conjugate, CAR, Tcell expressing a CAR, or nucleic acid molecule encoding such molecules,should provide a sufficient quantity of at least one of theIL-7Rα-specific antibody, antigen binding fragment, conjugate, CAR, Tcell expressing a CAR, or nucleic acid molecule encoding such moleculesto effectively treat the patient. The dosage can be administered once,but may be applied periodically until either a therapeutic result isachieved or until side effects warrant discontinuation of therapy. Inone example, a dose of the antibody or antigen binding fragment isinfused for thirty minutes every other day. In this example, about oneto about ten doses can be administered, such as three or six doses canbe administered every other day. In a further example, a continuousinfusion is administered for about five to about ten days. The subjectcan be treated at regular intervals, such as monthly, until a desiredtherapeutic result is achieved. Generally, the dose is sufficient totreat or ameliorate symptoms or signs of disease without producingunacceptable toxicity to the patient.

Data obtained from cell culture assays and animal studies can be used toformulate a range of dosage for use in humans. The dosage normally lieswithin a range of circulating concentrations that include the ED₅₀, withlittle or minimal toxicity. The dosage can vary within this rangedepending upon the dosage form employed and the route of administrationutilized. The therapeutically effective dose can be determined from cellculture assays and animal studies.

In certain embodiments, the antibody or antigen binding fragment thatspecifically binds IL-7Rα, or conjugate thereof, or a nucleic acidmolecule or vector encoding such a molecule, or a composition includingsuch molecules, is administered at a dose in the range of from about 5or 10 nmol/kg to about 300 nmol/kg, or from about 20 nmol/kg to about200 nmol/kg, or at a dose of about 5, 10, 15, 20, 25, 30, 35, 40, 45,50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 125, 130, 140,150, 160, 170, 175, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270,280, 290, 300, 350, 400, 450, 500, 750, 1000, 1250, 1500, 1750 or 2000nmol/kg, or at a dose of about 5, 10, 20, 30, 40, 50, 60, 70, 80, 90,100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 250, 300, 350,400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950 or 1000μg/kg, or about 1, 1.25, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7,7.5, 8, 8.5, 9, 9.5 or 10 mg/kg, or other dose deemed appropriate by thetreating physician. The doses described herein can be administeredaccording to the dosing frequency/frequency of administration describedherein, including without limitation daily, 2 or 3 times per week,weekly, every 2 weeks, every 3 weeks, monthly, etc.

In some embodiments, a disclosed therapeutic agent is administered maybe administered intravenously, subcutaneously or by another mode dailyor multiple times per week for a period of time, followed by a period ofno treatment, then the cycle is repeated. In some embodiments, theinitial period of treatment (e.g., administration of the therapeuticagent daily or multiple times per week) is for 3 days, 1 week, 2 weeks,3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks,11 weeks or 12 weeks. In a related embodiment, the period of notreatment lasts for 3 days, 1 week, 2 weeks, 3 weeks or 4 weeks. Incertain embodiments, the dosing regimen of the therapeutic agent isdaily for 3 days followed by 3 days off; or daily or multiple times perweek for 1 week followed by 3 days or 1 week off; or daily or multipletimes per week for 2 weeks followed by 1 or 2 weeks off; or daily ormultiple times per week for 3 weeks followed by 1, 2 or 3 weeks off; ordaily or multiple times per week for 4, 5, 6, 7, 8, 9, 10, 11 or 12weeks followed by 1, 2, 3 or 4 weeks off.

3. Modes of Administration

An IL-7Rα-specific antibody, antigen binding fragment, conjugate, CAR, Tcell expressing a CAR, or nucleic acid molecule encoding such molecules,or a composition including such molecules, as well as additional agents,can be administered to subjects in various ways, including local andsystemic administration, such as, e.g., by injection subcutaneously,intravenously, intra-arterially, intraperitoneally, intramuscularly,intradermally, or intrathecally. In an embodiment, a therapeutic agentis administered by a single subcutaneous, intravenous, intra-arterial,intraperitoneal, intramuscular, intradermal or intrathecal injectiononce a day. The therapeutic agent can also be administered by directinjection at or near the site of disease.

The therapeutic agent may also be administered orally in the form ofmicrospheres, microcapsules, liposomes (uncharged or charged (e.g.,cationic)), polymeric microparticles (e.g., polyamides, polylactide,polyglycolide, poly(lactide-glycolide)), microemulsions, and the like.

A further method of administration is by osmotic pump (e.g., an Alzetpump) or mini-pump (e.g., an Alzet mini-osmotic pump), which allows forcontrolled, continuous and/or slow-release delivery of the therapeuticagent or pharmaceutical composition over a pre-determined period. Theosmotic pump or mini-pump can be implanted subcutaneously, or near atarget site.

It will be apparent to one skilled in the art that the therapeutic agentor compositions thereof can also be administered by other modes.Determination of the most effective mode of administration of thetherapeutic agent or compositions thereof is within the skill of theskilled artisan. The therapeutic agent can be administered aspharmaceutical formulations suitable for, e.g., oral (including buccaland sub-lingual), rectal, nasal, topical, pulmonary, vaginal orparenteral (including intramuscular, intraarterial, intrathecal,subcutaneous and intravenous) administration, or in a form suitable foradministration by inhalation or insufflation. Depending on the intendedmode of administration, the pharmaceutical formulations can be in theform of solid, semi-solid or liquid dosage forms, such as tablets,suppositories, pills, capsules, powders, liquids, suspensions,emulsions, creams, ointments, lotions, and the like. The formulationscan be provided in unit dosage form suitable for single administrationof a precise dosage. The formulations comprise an effective amount of atherapeutic agent, and one or more pharmaceutically acceptableexcipients, carriers and/or diluents, and optionally one or more otherbiologically active agents.

4. Compositions

Compositions are provided that include one or more of theIL-7Rα-specific antibody, antigen binding fragment, conjugate, CAR, Tcell expressing a CAR, or nucleic acid molecule encoding such molecules,that are disclosed herein in a carrier. The compositions are useful, forexample, for example, for the treatment or prevention of ALL (such asT-ALL or B-ALL) in a subject. The compositions can be prepared in unitdosage forms for administration to a subject. The amount and timing ofadministration are at the discretion of the treating physician toachieve the desired purposes. The IL-7Rα-specific antibody, antigenbinding fragment, conjugate, CAR, T cell expressing a CAR, or nucleicacid molecule encoding such molecules can be formulated for systemic orlocal administration. In one example, the IL-7Rα-specific antibody,antigen binding fragment, conjugate, CAR, T cell expressing a CAR, ornucleic acid molecule encoding such molecules, is formulated forparenteral administration, such as intravenous administration.

In some embodiments, the compositions comprise an antibody, antigenbinding fragment, or conjugate thereof, in at least 70% (such as atleast 75%, at least 80%, at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98% or at least 99% purity. In certainembodiments, the compositions contain less than 10% (such as less than5%, less than 4%, less than 3%, less than 2%, less than 1%, less than0.5%, or even less) of macromolecular contaminants, such as othermammalian (e.g., human) proteins.

The compositions for administration can include a solution of theIL-7Rα-specific antibody, antigen binding fragment, conjugate, CAR, Tcell expressing a CAR, or nucleic acid molecule encoding such molecules,dissolved in a pharmaceutically acceptable carrier, such as an aqueouscarrier. A variety of aqueous carriers can be used, for example,buffered saline and the like. These solutions are sterile and generallyfree of undesirable matter. These compositions may be sterilized byconventional, well known sterilization techniques. The compositions maycontain pharmaceutically acceptable auxiliary substances as required toapproximate physiological conditions such as pH adjusting and bufferingagents, toxicity adjusting agents and the like, for example, sodiumacetate, sodium chloride, potassium chloride, calcium chloride, sodiumlactate and the like. The concentration of antibody in theseformulations can vary widely, and will be selected primarily based onfluid volumes, viscosities, body weight and the like in accordance withthe particular mode of administration selected and the subject's needs.

A typical composition for intravenous administration includes about 0.01to about 30 mg/kg of antibody or antigen binding fragment or conjugateper subject per day (or the corresponding dose of a conjugate includingthe antibody or antigen binding fragment). Actual methods for preparingadministrable compositions will be known or apparent to those skilled inthe art and are described in more detail in such publications asRemington's Pharmaceutical Science, 19th ed., Mack Publishing Company,Easton, Pa. (1995). In some embodiments, the composition can be a liquidformulation including one or more antibodies, antigen binding fragments(such as an antibody or antigen binding fragment that specifically bindsto IL-7Rα), in a concentration range from about 0.1 mg/ml to about 20mg/ml, or from about 0.5 mg/ml to about 20 mg/ml, or from about 1 mg/mlto about 20 mg/ml, or from about 0.1 mg/ml to about 10 mg/ml, or fromabout 0.5 mg/ml to about 10 mg/ml, or from about 1 mg/ml to about 10mg/ml.

Antibodies, or an antigen binding fragment thereof or a conjugate or anucleic acid encoding such molecules, can be provided in lyophilizedform and rehydrated with sterile water before administration, althoughthey are also provided in sterile solutions of known concentration. Theantibody solution, or an antigen binding fragment or a nucleic acidencoding such antibodies or antibody binding fragments, can then beadded to an infusion bag containing 0.9% sodium chloride, USP, andtypically administered at a dosage of from 0.5 to 15 mg/kg of bodyweight. Considerable experience is available in the art in theadministration of antibody drugs, which have been marketed in the U.S.since the approval of RITUXAN® in 1997. Antibodies, antigen bindingfragments, conjugates, or a nucleic acid encoding such molecules, can beadministered by slow infusion, rather than in an intravenous push orbolus. In one example, a higher loading dose is administered, withsubsequent, maintenance doses being administered at a lower level. Forexample, an initial loading dose of 4 mg/kg may be infused over a periodof some 90 minutes, followed by weekly maintenance doses for 4-8 weeksof 2 mg/kg infused over a 30 minute period if the previous dose was welltolerated.

Controlled-release parenteral formulations can be made as implants, oilyinjections, or as particulate systems. For a broad overview of proteindelivery systems see, Banga, A. J., Therapeutic Peptides and Proteins:Formulation, Processing, and Delivery Systems, Technomic PublishingCompany, Inc., Lancaster, Pa., (1995). Particulate systems includemicrospheres, microparticles, microcapsules, nanocapsules, nanospheres,and nanoparticles. Microcapsules contain the therapeutic protein, suchas a cytotoxin or a drug, as a central core. In microspheres thetherapeutic is dispersed throughout the particle. Particles,microspheres, and microcapsules smaller than about 1 μm are generallyreferred to as nanoparticles, nanospheres, and nanocapsules,respectively. Capillaries have a diameter of approximately 5 μm so thatonly nanoparticles are administered intravenously. Microparticles aretypically around 100 μm in diameter and are administered subcutaneouslyor intramuscularly. See, for example, Kreuter, J., Colloidal DrugDelivery Systems, J. Kreuter, ed., Marcel Dekker, Inc., New York, N.Y.,pp. 219-342 (1994); and Tice & Tabibi, Treatise on Controlled DrugDelivery, A. Kydonieus, ed., Marcel Dekker, Inc. New York, N.Y., pp.315-339, (1992).

Polymers can be used for ion-controlled release of the antibodycompositions disclosed herein. Various degradable and nondegradablepolymeric matrices for use in controlled drug delivery are known in theart (Langer, Accounts Chem. Res. 26:537-542, 1993). For example, theblock copolymer, polaxamer 407, exists as a viscous yet mobile liquid atlow temperatures but forms a semisolid gel at body temperature. It hasbeen shown to be an effective vehicle for formulation and sustaineddelivery of recombinant interleukin-2 and urease (Johnston et al.,Pharm. Res. 9:425-434, 1992; and Pec et al., J. Parent. Sci. Tech.44(2):58-65, 1990). Alternatively, hydroxyapatite has been used as amicrocarrier for controlled release of proteins (Ijntema et al., Int. J.Pharm.112:215-224, 1994). In yet another aspect, liposomes are used forcontrolled release as well as drug targeting of the lipid-capsulateddrug (Betageri et al., Liposome Drug Delivery Systems, TechnomicPublishing Co., Inc., Lancaster, Pa. (1993)). Numerous additionalsystems for controlled delivery of therapeutic proteins are known (seeU.S. Pat. No. 5,055,303; U.S. Pat. No. 5,188,837; U.S. Pat. No.4,235,871; U.S. Pat. No. 4,501,728; U.S. Pat. No. 4,837,028; U.S. Pat.No. 4,957,735; U.S. Pat. No. 5,019,369; U.S. Pat. No. 5,055,303; U.S.Pat. No. 5,514,670; U.S. Pat. No. 5,413,797; U.S. Pat. No. 5,268,164;U.S. Pat. No. 5,004,697; U.S. Pat. No. 4,902,505; U.S. Pat. No.5,506,206; U.S. Pat. No. 5,271,961; U.S. Pat. No. 5,254,342 and U.S.Pat. No. 5,534,496).

5. Methods of Detection and Diagnosis

Methods are also provided for the detection of the expression of IL-7Rαin vitro or in vivo. In one example, expression of IL-7Rα is detected ina biological sample, and can be used to detect the presence of a cellwith cell-surface expression of IL-7Rα in the sample. The sample can beany sample, including, but not limited to, tissue from biopsies,autopsies and pathology specimens. Biological samples also includesections of tissues, for example, frozen sections taken for histologicalpurposes. Biological samples further include body fluids, such as blood,serum, plasma, sputum, spinal fluid or urine. The method of detectioncan include contacting a cell or sample, or administering to a subject,an antibody or antigen binding fragment that specifically binds toIL-7Rα, or conjugate there of (e.g. a conjugate including a detectablemarker) under conditions sufficient to form an immune complex, anddetecting the immune complex (e.g., by detecting a detectable markerconjugated to the antibody or antigen binding fragment.

One embodiment provides a method of determining if a subject has cancerby contacting a sample from the subject with a monoclonal antibody (orconjugate) disclosed herein; and detecting binding of the antibody tothe sample. An increase in binding of the antibody to the sample ascompared to binding of the antibody to a control sample identifies thesubject as having cancer.

Another embodiment provides a method of confirming a diagnosis of cancerin a subject by contacting a sample from a subject diagnosed with cancerwith a monoclonal antibody (or conjugate) disclosed herein; anddetecting binding of the antibody to the sample. An increase in bindingof the antibody to the sample as compared to binding of the antibody toa control sample confirms the diagnosis of cancer in the subject.

In some embodiments, the cancer is ALL, such as T-ALL or B-ALL. In someembodiments the cancer is one that expresses IL-7Rα. In some examples,the control sample is a sample from a subject without cancer. Inparticular examples, the sample is a blood or tissue sample.

In one embodiment, the antibody or antigen binding fragment is directlylabeled with a detectable marker. In another embodiment, the antibodythat binds IL-7Rα (the first antibody) is unlabeled and a secondantibody or other molecule that can bind the antibody that binds thefirst antibody is utilized for detection. As is well known to one ofskill in the art, a second antibody is chosen that is able tospecifically bind the specific species and class of the first antibody.For example, if the first antibody is a human IgG, then the secondaryantibody may be an anti-human-IgG. Other molecules that can bind toantibodies include, without limitation, Protein A and Protein G, both ofwhich are available commercially.

Suitable labels for the antibody, antigen binding fragment or secondaryantibody are described above, and include various enzymes, prostheticgroups, fluorescent materials, luminescent materials, magnetic agentsand radioactive materials. Non-limiting examples of suitable enzymesinclude horseradish peroxidase, alkaline phosphatase,beta-galactosidase, or acetylcholinesterase. Non-limiting examples ofsuitable prosthetic group complexes include streptavidin/biotin andavidin/biotin. Non-limiting examples of suitable fluorescent materialsinclude umbelliferone, fluorescein, fluorescein isothiocyanate,rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride orphycoerythrin. A non-limiting exemplary luminescent material is luminol;a non-limiting exemplary a magnetic agent is gadolinium, andnon-limiting exemplary radioactive labels include ¹²⁵I, ¹³¹I, ₃₅ S or³H.

F. Kits

Kits are also provided. For example, kits for treating a subject with acancer that expresses IL-7Rα, or for detecting IL-7Rα in a sample or ina subject. The kits will typically include a disclosed IL-7Rα-specificantibody, antigen binding fragment, conjugate, CAR, T cell expressing aCAR, or nucleic acid molecule encoding such molecules, or compositionsincluding such molecules.

In one embodiment, the kit is a diagnostic kit and includes animmunoassay. Although the details of the immunoassays may vary with theparticular format employed, the method of detecting IL-7Rα in abiological sample generally includes the steps of contacting thebiological sample with an antibody which specifically reacts, underconditions sufficient to form an immune complex, to IL-7Rα. The antibodyis allowed to specifically bind under immunologically reactiveconditions to form an immune complex, and the presence of the immunecomplex (bound antibody) is detected directly or indirectly.

The kit can include a container and a label or package insert on orassociated with the container. Suitable containers include, for example,bottles, vials, syringes, etc. The containers may be formed from avariety of materials such as glass or plastic. The container typicallyholds a composition including one or more of the disclosed antibodies,antigen binding fragments, conjugates, nucleic acid molecules, orcompositions. In several embodiments the container may have a sterileaccess port (for example the container may be an intravenous solutionbag or a vial having a stopper pierceable by a hypodermic injectionneedle). A label or package insert indicates that the composition isused for treating the particular condition.

The label or package insert typically will further include instructionsfor use of the antibodies, antigen binding fragments, conjugates,nucleic acid molecules, or compositions included in the kit. The packageinsert typically includes instructions customarily included incommercial packages of therapeutic products that contain informationabout the indications, usage, dosage, administration, contraindicationsand/or warnings concerning the use of such therapeutic products. Theinstructional materials may be written, in an electronic form (such as acomputer diskette or compact disk) or may be visual (such as videofiles). The kits may also include additional components to facilitatethe particular application for which the kit is designed. Thus, forexample, the kit may additionally contain means of detecting a label(such as enzyme substrates for enzymatic labels, filter sets to detectfluorescent labels, appropriate secondary labels such as a secondaryantibody, or the like). The kits may additionally include buffers andother reagents routinely used for the practice of a particular method.Such kits and appropriate contents are well known to those of skill inthe art.

IV. EXAMPLES

The following examples are provided to illustrate particular features ofcertain embodiments, but the scope of the claims should not be limitedto those features exemplified.

Example 1 IL-7Rα-Specific Antibodies for Treating ALL

This example illustrates the isolation and characterization of the 4A10and 2B8 antibodies. These antibodies specifically bind to theextracellular domain of IL-7Rα, and can direct ADCC-mediated killing ofcells the express IL-7Rα on their surface. Further, an in vivo model ofT-ALL is used to show that a chimeric form of the 4A10 antibodyincluding a human constant region can direct ADCC-mediated killing ofALL cells (including T-ALL cells) expressing a gain-of-function mutantform of the IL-7R that drives cell proliferation.

Introduction

Acute lymphoblastic leukemia (ALL) is the most common cancer inchildren, with approximately 3250 new cases per year in the UnitedStates. Treatment for ALL has improved dramatically in recent decades,but there remain about 20% of cases that are not cured and ALL remains aleading cause of death in children. ALL also occurs in adults where ithas a far less favorable prognosis. The need for new therapeutics in ALLis illustrated, for example, in the incidence of cognitive impairmentwith chemotherapy in the developing brain (Cheung and Krull, Neurosci.Biobehay. Rev., 53:108-120, 2015). ALL can be subdivided into two broadgroups: those derived from immature T cells (T-ALL) and those derivedfrom immature B cells (B-ALL). There have been great advances inunderstanding the genetic basis of ALL, leading to newsub-classifications of patients based on their genetic aberrationscombined with surface phenotype (Mullighan, J. Clinical Invest.,122:3407-3415, 2012; Van Vlierberghe and Ferrando, J. Clinical Invest.,122:3398-3406, 2012).

IL-7, a product of stromal cells, is normally required for T and B celldevelopment and for survival of mature T cells. IL-7 acts on lymphocytesby binding with high affinity to IL-7Rα, then recruiting the common γcchain. This heterodimerization brings together the intracellular domainsof IL-7Rα and γc and their associated kinases, Jak1 and Jak3respectively. These tyrosine kinases have a low level of intrinsicsignaling activity, which is greatly increased by mutualphosphorylation. Jak1 and Jak3 then phosphorylate a site on theintracellular domain of IL-7Rα, which recruits Stat5. Stat5 is thenphosphorylated, inducing its dimerization and dissociation from IL-7Rαand translocation to the nucleus where it serves as a transcriptionfactor, inducing genes involved in survival and proliferation (reviewedin Jiang et al., Cytokine Growth Factor Rev., 16:513-533, 2005, which isincorporated by reference herein). Most of the lymphocyte requirementfor IL-7 is attributable to these survival and proliferative effects,which also makes the IL-7 pathway potentially vulnerable to mutationscausing cancer.

Prior findings identified gain-of-function mutations in the IL-7Rα genethat lead to over production and accumulation of T-ALL cells (Zenatti etal., Nat. Genet., 43:932-939, 2011, which is incorporated by referenceherein). These prior studies found that mutations are observed inmultiple cohorts of T-ALL in about 10% of patients (see FIG. 1). Thesemutations are typically insertions containing cysteines into exon 6 ofthe IL-7Rα gene, which encodes the border of theextracellular/transmembrane domains of the IL-7Rα protein (see FIG. 2,and, e.g., Shochat et al., J. Exp. Med., 208:901-908, 2011; and Zhang etal., Nature, 481:157-163, 2012, each of which is incorporated byreference herein). Other gain-of-function mutations in the IL-7 pathwaycan also occur in T-ALL (such as gain-of-function mutations in the genesencoding IL-7Rα, Jak1, Jak3, Stat5b, or Ras), as well as in B-ALL (suchas gain-of-function mutations in the genes encoding TSLPR or Jak2) (seeFIG. 3). Thus, the IL-7 receptor pathway contains vulnerableproto-oncogenes for immature lymphocytes and targeting this pathwayoffers therapeutic potential in ALL. Further, the majority of T-ALLcells express IL-7Rα and respond to IL-7 in vitro, showing increasedsurvival (Barata et al., Blood, 98:1524-1531, 2001; Touw et al., Blood,75:2097-2101, 1990). Accordingly, monoclonal antibodies that targetIL-7Rα may be effective against T-ALL with mutant or WT IL-7Rα. AsIL-7Rα can be present on the cell-surface of B cells associated withB-ALL, IL-7Rα-directed antibody may also be effective therapeutics forB-ALL.

Several monoclonal antibodies have been used clinically to treat cancersand their mechanisms of action differ widely. In some examples,monoclonal antibodies are shown to block the signaling from a receptor,whereas in other examples, monoclonal antibodies target cancer cells forkilling by innate immune cells. Anti-CD20 (Rituximab) is widely used totreat non-Hodgkin lymphoma and B cell chronic lymphocytic leukemia.Based on its effect of eliminating normal B cells, anti-CD20 is alsoused to treat psoriasis. Anti-CD20 is thought to mediate its effects viaADCC and phagocytosis of lymphoma and normal B cells (Wilson et al.,Cancer Cell, 19:101-113, 2011), and it may enhance presentation of tumorantigens to the immune system (reviewed in Abes and Teillaud, CancerMetastasis Rev., 30:111-124, 2011). In treatment of breast cancer,anti-Her2 (Trastuzumab) juxtamembrane region induces both uncouplingfrom Her3 as well as ADCC (reviewed in Garrett and Arteaga, Cancer Biol.Ther., 11:793-800, 2011). In treatment of squamous cell carcinoma andcolon cancer, the mechanism of anti-EGFR (Cetuximab) is based onblocking receptor dimerization as well as ADCC (reviewed in Brand etal., Cancer Biol. Ther., 11:777-792, 2011). Accordingly, ALL cells inpatients may be killed by IL-7Ra targeted monoclonal antibodies based onseveral possible mechanisms ranging from blocked signaling to targetingcells for ADCC, and we recognize that, analogous to anti-CD20, therecould be killing of the patient's normal T cells.

Isolation and Characterization of the 4A10 and 2B8 Antibodies

Mouse monoclonal antibodies directed against IL-7Rα were generated usingstandard hybridoma production assays. An engineered IL-7Rα extracellulardomain from a T-ALL patient that encoded a gain-of-function cysteineinsertion was expressed in insect cells, and the resulting solubleIL-7Rα ectodomain homodimer was purified. Mice were immunized with thepurified soluble T-ALL IL-7Rα homodimer, and antibody produced fromhybridoma cell lines prepared from the immunized mice was screened forIL-7Rα binding. The first round of screenings was performed on WT IL-7Rαextracellular domain immobilized on plastic, and although several cloneswere found, none bound to IL-7Rα on the cell surface. Accordingly, asecond round of screening was performed to assay for mAb that binds tocells expressing full-length WT IL-7Rα. Two positive hybridomas wereidentified that resulted in 4A10 and 2B8 antibodies, respectively. Theidentified antibodies, and corresponding sequence identifiers are asfollows:

V_(H) V_(L) CDR V_(L) V_(L) CDR V_(H) V_(L) protein (kabat) protein(Kabat) DNA DNA Antibody SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID 4A101 5, 6, 7 2 8, 9, 10 25 26 2B8 3 11, 12, 13 4 14, 15, 16 27 28

The binding affinity of the 4A10 and 2B8 antibodies for IL-7Rα wasevaluated using surface plasmon resonance (FIG. 4). scFvs including theheavy and light chain variable regions of the 4A10 or 2B8 antibody wereprepared and used in the binding assays. IL-7Rα ectodomain was coupledto an SPR sensor-chip and binding was assayed by injecting 4A10 or 2B8scFv over the flow cells of the sensor-chip, and detecting the bindingresponse. As illustrated in FIG. 4, the calculated K_(D) for 2B8 and4A10 binding to IL-7Rα is 8.7 nM and 3.7 nM, respectively.

The 4A10 and 2B8 epitopes on IL-7Rα were further evaluated using surfaceplasmon resonance (FIG. 5). scFvs including the heavy and light chainvariable regions of the 4A10 and 2B8 antibodies were prepared and usedin the binding assays. The WT IL-7Rα ectodomain was coupled to the SPRsensor-chip and binding was assayed by injecting 100 μL (400 mM) of 4A10or 2B8 scFv over the sensor-chip, followed by a sequential injection ofanother 100 μL (400 μM) of 4A10 or 2B8 scFv. The dashed line in FIG. 5indicates the time point when the second scFv solution was injected. Anincreased response was observed when 2B8 injection was followed by 4A10injection, indicating that these two scFvs bind to non-overlappingepitopes on IL-7Rα.

Additional binding studies were performed to show that the 4A10 and 2B8antibodies bind to IL-7Rα on the cell surface. D1 cells expressingeither wild-type human IL-7Rα or mutant human IL-7Rα that includes theP1 mutation were incubated with chimeric antibody including the heavyand light chain variable regions of 4A10 or 2B8, and a human IgG1constant region, and bound antibody was detected using FACS analysis. Asillustrated by the following table, this study shows that the chimeric4A10 and 2B8 antibodies (in IgG1 format) each bind to IL-7Rα on thecell-surface, and bind to different epitopes on the IL-7Rα ectodomain.

Binding to IL-7Rα expressed on D1 cells (arbitrary units) mAb D1-hIL-7RαWT D1-hIL-7Rα P1 2B8-hIgG1 chimera 71.1 65.5 4A10-hIgG1 chimera 76.477.0 2B8-hIgG1 chimera + 171.5 149.9 4A10-hIgG1 chimera

Additional surface plasmon resonance binding studies were performed toevaluate the binding of the 4A10 and 2B8 antibodies to glycosylated andunglycosylated forms of the IL-7Rα ectodomain. The Fab fragment of the4A10-hIgG1 chimera was generated using papain cleavage subsequentlypurified. Binding constants (K_(D)) of 9.2 nM and 91.2 nM were measuredfor binding of the Fab fragment of the 4A10 MAb to glycosylated andunglycosylated forms of the IL-7Rα ectodomain, respectively. Bindingconstants (K_(D)) of 1.1 nM and 19.8 nM were measured for binding of thefull-length 4A10-hIgG1 chimera to glycosylated and unglycosylated formsof the IL-7Rα ectodomain, respectively. Accordingly, the 4A10 antibodycan recognize different glycosylation states of the IL-7Rα, which may beuseful as a diagnostic reagent for tissues and fluids.

Further, the 4A10 antibody was found to bind to human T cells thatexpress human IL-7Rα with and without the “P1” or “P7” gain-of-functionmutation (FIG. 6). Additionally, competition assays showed that the 4A10and 2B8 antibodies do not compete with IL-7 for binding to the IL-7R,and therefore bind to epitopes that do not overlap with the IL-7 bindingsite on IL-7R.

Additionally, the 4A10 antibody binds to human T-ALL cells (FIG. 7). AT-ALL patient sample was transplanted into immunodeficient (NSG) mice.Spleen cells were harvested from the mice, and assayed for 4A10-hIgG1chimera binding using FACS. The spleen cells were held overnight at 4°C. and blocked with 0.5 μl of 2.4G2 for 20 minutes prior to staining andFACS analysis. Block was present for primary antibody incubation. The4A10-hIgG1 chimera also stained T cells from normal human blood andmacaque blood, as evaluated using FACS binding assays.

A 4A10-hIgG1 Chimera Mediates ADCC killing of IL-7Rα Positive CancerCells In Vitro

An important mechanism for killing leukemia cells can be ADCC, asillustrated by anti-CD20 killing of lymphoma cells. Accordingly, the4A10-hIgG1 chimera was evaluated for ADCC-mediated killing of IL-7Rαexpressing cells (FIG. 8). BaF3 cells (a murine B cell line that doesnot express IL-7Rα) were incubated with NK cells isolated from humanblood and the 4A10-hIgG1 chimera (at 10 μg/ml). LDH release was measuredto evaluate cell lysis (killing). As shown in FIG. 8, the 4A10-hIgG1antibody effectively mediated NK-cell lysis of the BaF3 cells in this invitro assay. Similar results were obtained using the D1 cells (an IL-7dependent thymocyte cell line) (FIG. 9). Additionally, the 4A10-hIgG1chimera mediated NK-cell directed killing of T-ALL cells expressing P1gain-of-function IL-7Rα (FIG. 10) and normal human T cells (FIG. 11).

A 4A10-hIgG Chimera Reduces Tumor Burden and Prolongs Survival in InVivo Models of ALL

To evaluate the therapeutic effect of the 4A10-hIgG1 chimera in vivo, aleukemia model was used (FIG. 12). Mutant IL-7Rα P1 was transfectedtogether with GFP into D1 cells (creating a murine lymphoid cell linethat grows and metastasizes in mice) and Rag −/− mice were inoculatedwith the transfected cells (this model was previously described inZenatti et al., Nat. Genet., 43:932-939, 2011). The day after leukemiccell inoculation, the 4A10-hIgG1 chimera was administered to the mice atvarious doses. Blood and tissue were sampled at various time pointpost-inoculation, and the overall survival of the mice was alsoevaluated. A diagram illustrating the procedure is provided in FIG. 12A.A dramatic reduction in leukemic cells was observed in samples taken onday 15 from spleen (FIG. 12B), bone marrow (FIG. 12C), and liver (FIG.12D) in mice administered 250 μg 4A10-hIgG1 chimera by IV compared tothe PBS control. FIG. 12E provide a summary of leukemic cell number inspleen and liver at day 19 from various experimental conditions.Finally, as shown in FIG. 12F, a single injection of the 4A10-hIgG1chimera prolonged survival of mice inoculated with the IL-7Rαtransformed D1 cells. Control mice not receiving MAb died on day 17 and18.

To further evaluate the therapeutic effect of the 4A10-hIgG1 chimera invivo, an additional leukemia model was used (FIG. 13). Immunodeficient(NSG) mice were inoculated with T-ALL cells with a WT IL-7Rα gene thatwere harvested from a human patient (5×10⁶ were administered IV). NSGmice lack NK, T and B cells. The 4A10-hIgG1 chimera (400 μg) wasadministered to the mice intravenously at weekly intervals totaling fiveinjections. Blood and tissue were sampled at various time pointpost-inoculation, and the overall survival of the mice was alsoevaluated. A diagram illustrating the procedure is provided in FIG. 13A.A dramatic reduction in T cells (detected by human IL-7R and CD4staining) was observed in samples taken on day 33 from spleen (FIG.13B), blood (FIG. 13C) and liver (FIG. 13D), but depletion wasincomplete in NSG bone marrow samples, from mice treated with the4A10-hIgG1 compared to the PBS control. Surprisingly, the Mab showedefficacy in controlling human T-ALL growth, in the absence of NK cells,at multiple sites (FIG. 13). However, bone marrow harbored viable T-ALLcells in treated NSG mice.

Additional assays were performed to show that NK cells promote theefficacy of anti-IL-7R treatment of T-ALL. These assays were performedto evaluate use of the 4A10-hIgG1 chimeric mAb to reduce xenograftedhuman T-ALL cells in the presence of NK cells. The assays used NOD/Scidmice, which have NK cells but are deficient in T and B cells. Miceharboring human T-ALL cells were treated with weekly injections of the4A10-hIgG1 chimeric mAb (FIG. 14A). Treatment was highly effective inblood, liver, lung, and most importantly bone marrow (FIGS. 14B-14E),the latter site having shown resistance to treatment in the absence ofNK cells. Further, a significant increase in survival with the mAbtreatment was observed (FIG. 14F).

IL-7Rα Antibodies Inhibit IL-7 Signaling in T-ALL Cells

Additional in vitro assays were performed to show that chimeric mAbshave inhibitory effects on IL-7-induced signaling. T-ALL cells respondto IL-7 and induce phosphorylation of STAT5, an early signalingmolecule. For these assays, T-ALL #5 cells were isolated from engraftedNSG spleen. The cells cultured with anti-IL-7Rα antibody for 2 hours at4C, followed by stimulated with hIL-7 for 20 minutes and evaluationusing flow cytometry (ICFC) for pSTAT-5. The 4A10-hIgG1 chimeric mAbblocked IL-7-induced pSTAT5 response at the lower dose of IL-7 (FIG. 15,right panel) and partially blocked at the higher dose of IL-7 (FIG. 15,left panel). In contrast, the 2B8 mAb had no inhibitory activity on itsown, but cooperated with the 4A10-hIgG1 chimeric mAb to block signalingat the higher dose of IL-7 (FIG. 15, left panel). As noted above, 4A10and 2B8 bind non-overlapping epitopes on IL-7R. This data shows that4A10 alone inhibits IL-7R signaling at low IL-7 doses and cooperateswith 2B8 at high doses, suggesting future therapeutics could combine thetwo antibodies. Thus, one non-limiting explanation for the inhibitoryeffect of the 4A10-hIgG1 chimera on T-ALL in mice lacking NK cells isthat this effect is due to inhibiting survival signals from the lowlevels of IL-7 found in vivo.

Another approach to determine whether anti-IL-7R antibodies can inhibitT-ALL in vivo, independent of ADCC would be to test the effect of theoriginal mouse 4A10 antibodies. These contain the mouse IgG1 constantregion which cannot mediate ADCC, unlike the ADCC-mediating chimericantibodies. These experiments are ongoing, but so far there is asubstantial inhibitory effect of the original 4A10 mouse Mab on a T-ALLxenograft.

In summary, the data provided in this example illustrate the specificbinding activity of the 4A10 and 2B8 antibodies for IL-7Rα, and the invivo efficacy of the 4A10 antibody for treating leukemias that expressIL-7Rα, such as T-ALL.

Example 2 Combination Therapy with IL-7Rα Specific Monoclonal Antibodyand CXCR4 Antagonist Synergistically Depletes T-ALL Cells from BoneAarrow

This example illustrates that IL-7Rα mAb and CXCR4 antagonistcombination therapy synergistically reduces T-ALL cells from bonemarrow.

Prior studies showed that the C-X-C chemokine receptor type 4 isessential for leukemia-initiating cell (LIC) activity, including forT-ALL cells, and proposed CXCR4 antagonists for treatment of T-ALL (seee.g., Passaro et al., Cancer Cell, 27(6):769-779, 2015). To determine ifCXCR4 antagonists and IL-7Rα specific antibodies could act incombination to reduce T-ALL, combination therapy including the4A10-hIgG1 chimeric antibody and a CXCR4 antagonist (AMD3100) was testedin a T-ALL xenograft model using immunodeficient mice. The mice received10 mg/kg of AMD3100 subcutaneously Monday through Friday.

Surprisingly, combing the CXCR4 antagonist (AMD3100) together with4A10-hIgG1 chimeric antibody was highly effective in and synergisticallydepleted leukemia cells from bone marrow (see FIG. 16).

Example 3 IL-7Rα-Specific Monoclonal Antibodies for Detecting Cancer ina Subject or Confirming the Diagnosis of Cancer in a Subject

This example describes the use of IL-7Rα-specific monoclonal antibodies(such as an antibody with the CDRs of the 4A10 or 2B8 mAb as disclosedherein) for the detection of cancer in a subject. This example furtherdescribes the use of these antibodies to confirm the diagnosis of cancerin a subject.

A blood or tissue sample (such as a biopsy) is obtained from the patientdiagnosed with, or suspected of having an IL-7Rα-positive cancer (i.e.,a cancer that overexpresses IL-7Rα, or overexpresses IL-7Rα activity,such as ALL, for example, T-ALL or B-ALL). A sample taken from a patientthat does not have cancer can be used as a control. An immunoassay (suchas immunohistochemistry or fluorescence in situ hybridization of atissue sample) is performed to detect the presence of IL-7Rα in thesample (such as IL-7Rα-expressing cells in a tissue sample). Forexample, tissue sections obtained from a patient biopsy and a controltissue sample are contacted with an IL-7Rα-specific monoclonal antibodydirectly conjugated with a detectable label (such as an enzyme) andimmunohistochemical detection for IL-7Rα is carried out according tostandard procedures. An increase in enzyme activity of the patientsample, relative to the control sample, indicates the anti-IL-7Rαantibody specifically bound proteins from the tissue sample, thusdetecting the presence of IL-7Rα protein in the sample. Detection ofIL-7Rα protein in the patient sample indicates the patient has anIL-7Rα-positive cancer, or confirms diagnosis of cancer in the subject.

Example 4 Treatment of T-ALL in a Subject

This example describes a particular method that can be used to treat acancer that expresses IL-7Rα in humans by administration of a chimericantibody including the CDRs of the 4A10 or 2B8 antibody and/or the V_(H)and V_(L) of the 4A10 or 2B8 antibody, and a human IgG1 constant region.Although particular methods, dosages, and modes of administrations areprovided, one skilled in the art will appreciate that variations can bemade without substantially affecting the treatment.

In this example, patients diagnosed with T-ALL are administered thechimeric antibody. Preparation of the chimeric antibody is performedaccording to standard methods. In some patients, the chimeric antibodyis administered by intravenous infusion every three weeks. The dose ofthe chimeric antibody administered to a patient varies depending on theweight and gender of the patient, and mode and time course ofadministration. In some cases, the chimeric antibody is administered ata dose of about 1 to about 5 mg/kg. Following treatment, patients areevaluated for cancer progression (including cancer growth andmetastasis) and other clinical signs of illness.

It will be apparent that the precise details of the methods orcompositions described may be varied or modified without departing fromthe spirit of the described embodiments. We claim all such modificationsand variations that fall within the scope and spirit of the claimsbelow.

1. An isolated monoclonal antibody that specifically binds to anextracellular domain of IL-7Rα, comprising: a heavy chain variableregion (V_(H)) comprising a heavy chain complementarity determiningregion (HCDR)1, a HCDR2, and a HCDR3 of the V_(H) set forth as SEQ IDNO: 1 (4A10 V_(H)) and a light chain variable region (V_(L)) comprisinga light chain complementarity determining region (LCDR)1, a LCDR2, and aLCDR3 of the V_(L) set forth as SEQ ID NO: 2 (4A10 V_(L)); or a V_(H)comprising a HCDR1, a HCDR2, and a HCDR3 of the V_(H) set forth as SEQID NO: 3 (2B8 V_(H)) and a V_(L) comprising a LCDR1, a LCDR2, and aLCDR3 of the V_(L) set forth as SEQ ID NO: 4 (2B8[[4A10]] V_(L)).
 2. Theantibody of claim 1, wherein the HCDR1, the HCDR2, the HCDR3, the LCDR1,the LCDR2, and the LCDR3 comprise the amino acids sequences set forth asSEQ ID NOs: 5, 6, 7, 8, 9, and 10, respectively (4A10 kabat CDRs) or theamino acids sequences set forth as SEQ ID NOs: 11, 12, 13, 14, 15, and16, respectively (2B8 kabat CDRs).
 3. (canceled)
 4. The antibody ofclaim 1, wherein the V_(H) and V_(L) comprise the amino acid sequencesset forth as SEQ ID NOs: 1 and 2, respectively, or amino acid sequencesset forth as SEQ ID NOs: 3 and 4, respectively.
 5. (canceled)
 6. Theantibody of claim 1, comprising human framework regions and/or a humancontact region.
 7. (canceled)
 8. The antibody of claim 1, wherein theantibody is an IgG, IgM or IgA.
 9. The antibody of claim 1, wherein theantibody is an IgG1 and comprises a human constant region.
 10. Theantibody of claim 9, comprising a heavy chain and a light chaincomprising the amino acid sequences set forth as SEQ ID NOs: 21 and 22,respectively, or SEQ ID NOs: 23 and 24, respectively.
 11. The antibodyof claim 1, comprising a constant region comprising a modification thatincreases binding to the neonatal Fc receptor and/or increasesantibody-dependent cell cytotoxicity (ADCC).
 12. The antibody of claim1, wherein: the antibody mediates ADCC killing of IL-7Rα positive cells;and/or the antibody inhibits IL-7 signaling in IL-7Rα positive cells.13. (canceled)
 14. The antibody of claim 12, wherein the cells are T-ALLcells.
 15. An antigen binding fragment that specifically binds to theextracellular domain IL-7Rα, comprising the V_(H) and the V_(L) of theantibody of claim
 1. 16. The antigen binding fragment of claim 15,wherein the antigen binding fragment is a Fv, Fab, F(ab′)₂, scFV or ascFV₂ fragment.
 17. A bispecific antibody that specifically binds to theextracellular domain IL-7Rα, comprising the V_(H) and the V_(L) of theantibody of claim
 1. 44.
 18. The bispecific antibody of claim 17,wherein the antibody specifically binds to IL-7Rα and to CD3.
 19. Theantibody of claim 1, linked to an effector molecule or a detectablemarker
 20. (canceled)
 21. An antibody-drug conjugate comprising a drugconjugated to an antibody comprising the V_(H) and the V_(L) of theantibody of claim
 1. 22. The antibody-drug conjugate of claim 21,wherein the drug is a chemotherapeutic agent; and optionally wherein thechemotherapeutic agent is monomethyl auristatin E, monomethyl auristatinF, or DM1 and/or the drug is conjugated to the antibody by a cleavablepeptide linker. 23.-24. (canceled)
 25. An isolated nucleic acid moleculeencoding the V_(H) and/or the V_(L) of the antibody or antigen bindingfragment of claim
 1. 26. The nucleic acid molecule of claim 25, whereinthe V_(H) and the V_(L) of the antibody or antigen binding fragmentcomprise the nucleic acid sequences set forth as: SEQ ID NOs: 25 and 26,respectively, or degenerate variants thereof; or SEQ ID NOs: 27 and 28,respectively, or degenerate variants thereof.
 27. The nucleic acidmolecule of claim 25, encoding a chimeric antigen receptor comprising anextracellular domain comprising a scFv comprising the V_(H) and theV_(L) of the antibody or antigen binding fragment.
 28. The nucleic acidmolecule of claim 25, operably linked to a promoter.
 29. An expressionvector comprising the nucleic acid molecule of claim
 28. 30. An isolatedhost cell transformed with the nucleic acid molecule or expressionvector of claim
 25. 31. The host cell of claim 30, wherein the host cellis a T cell.
 32. A pharmaceutical composition, comprising atherapeutically effective amount of: the antibody of claim 1, an antigenbinding fragment of the antibody, a bispecific antibody or antibody-drugconjugate comprising the antibody or antigen binding fragment, a nucleicacid molecule encoding the antibody, antigen binding fragment, orbispecific antibody, or an expression vector or host cell comprising thenucleic acid molecule; and a pharmaceutically acceptable carrier.
 33. Amethod of treating a subject with an IL-7Rα-positive cancer, comprisingadministering to the subject a therapeutically effective amount of thecomposition of claim 32, thereby treating the cancer in the subject. 34.The method of claim 33, further comprising administering to the subjecta therapeutically effective amount of a C-X-C chemokine receptor type 4(CXCR4) antagonist.
 35. The method of claim 34, wherein the CXCR4antagonist is AMD3100.
 36. A method of determining if a subject has anIL-7Rα expressing cancer, comprising: contacting a sample from thesubject with the antibody of claim 1; and detecting binding of theantibody or antigen binding fragment to the sample, wherein an increasein binding of the antibody to the sample as compared to binding of theantibody to a control sample identifies the subject as having the cancerthat expresses IL-7Rα. 37.-38. (canceled)
 39. The method of claim 33,wherein the IL-7Rα-positive cancer comprises a mutation in the IL-7pathway that increases proliferation of lymphocytes, optionally whereinthe mutation is a gain-of-function mutation in the gene encoding IL-7Rαthat leads to increased phosphorylation of Stat5b compared to control.40. (canceled)
 41. The method of claim 33, wherein the cancer is anacute lymphoblastic leukemia (ALL), optionally wherein the ALL is B-ALLor T-ALL. 42.-43. (canceled)
 44. A method of treating or preventing anautoimmune disease in a subject, comprising: administering atherapeutically effective amount of the antibody of claim 1 to thesubject. 45.-48. (canceled)